Prevention and treatment of amyloidogenic disease

ABSTRACT

The invention provides compositions and methods for treatment of amyloidogenic diseases. Such methods entail administering an agent that induces a beneficial immune response against an amyloid deposit in the patient. The methods are particularly useful for prophylactic and therapeutic treatment of Alzheimer&#39;s disease. In such methods, a suitable agent is Aβ peptide, active fragments thereof or an antibody thereto.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.09/724,319, filed Nov. 27, 2000, which is a continuation of U.S.application Ser. No. 09/322,289, filed May 28, 1999, which is acontinuation-in-part of U.S. application Ser. No. 09/201,430, filed Nov.30, 1998 which claims the benefit under 35 U.S.C. 119(e) of U.S.Application No. 60/080,970, filed Apr. 7, 1998, and U.S. Application60/067,740, filed Dec. 2, 1997, all of which are incorporated byreference in their entirety for all purposes.

TECHNICAL FIELD

The invention resides in the technical fields of immunology andmedicine.

BACKGROUND OF THE INVENTION

Alzheimer's disease (AD) is a progressive disease resulting in seniledementia. See generally Selkoe, TINS 16, 403-409 (1993); Hardy et al.,WO 92/13069; Selkoe, J. Neuropathol. Exp. Neurol. 53, 438-447 (1994);Duff et al., Nature 373, 476-477 (1995); Games et al., Nature 373, 523(1995). Broadly speaking the disease falls into two categories: lateonset, which occurs in old age (65+ years) and early onset, whichdevelops well before the senile period, i.e, between 35 and 60 years. Inboth types of disease, the pathology is the same but the abnormalitiestend to be more severe and widespread in cases beginning at an earlierage. The disease is characterized by at least two types of lesions inthe brain, senile plaques and neurofibrillary tangles. Senile plaquesare areas of disorganized neuropil up to 150 μm across withextracellular amyloid deposits at the center visible by microscopicanalysis of sections of brain tissue. Neurofibrillary tangles areintracellular deposits of microtubule associated tau protein consistingof two filaments twisted about each other in pairs.

The principal constituent of the plaques is a peptide termed Aβ orβ-amyloid peptide. Aβ peptide is an internal fragment of 39-43 aminoacids of a precursor protein termed amyloid precursor protein (APP).Several mutations within the APP protein have been correlated with thepresence of Alzheimer's disease. See, e.g., Goate et al., Nature 349,704) (1991) (valine⁷¹⁷ to isoleucine); Chartier Harlan et al. Nature353, 844 (1991)) (valine⁷¹⁷ to glycine); Murrell et al., Science 254, 97(1991) (valine⁷¹⁷ to phenylalanine); Mullan et al., Nature Genet. 1, 345(1992) (a double mutation changing lysine⁵⁹⁵-methionine⁵⁹⁶ toasparagine⁵⁵⁹-leucine⁵⁹⁶). Such mutations are thought to causeAlzheimer's disease by increased or altered processing of APP to Aβ,particularly processing of APP to increased amounts of the long form ofAβ (i.e., Aβ1-42 and Aβ1-43). Mutations in other genes, such as thepresenilin genes, PS1 and PS2, are thought indirectly to affectprocessing of APP to generate increased amounts of long form Aβ (seeHardy, TINS 20, 154 (1997)). These observations indicate that Aβ, andparticularly its long form, is a causative element in Alzheimer'sdisease.

McMichael, EP 526,511, proposes administration of homeopathic dosages(less than or equal to 10⁻² mg/day) of Aβ to patients withpreestablished AD. In a typical human with about 5 liters of plasma,even the upper limit of this dosage would be expected to generate aconcentration of no more than 2 pg/ml. The normal concentration of Aβ inhuman plasma is typically in the range of 50-200 pg/ml (Seubert et al.,Nature 359, 325-327 (1992)). Because EP 526,511's proposed dosage wouldbarely alter the level of endogenous circulating Aβ and because EP526,511 does not recommend use of an adjuvant, as an immunostimulant, itseems implausible that any therapeutic benefit would result.

By contrast, the present invention is directed inter alia to treatmentof Alzheimer's and other amyloidogenic diseases by administration of Aβ,other active immunogen or antibody to Aβ to a patient under conditionsthat generate a beneficial immune response in the patient. The inventionthus fulfills a longstanding need for therapeutic regimes for preventingor ameliorating the neuropathology and, in some patients, the cognitiveimpairment associated with Alzheimer's disease.

SUMMARY OF THE CLAIMED INVENTION

In one aspect, the invention provides methods of preventing or treatinga disease characterized by amyloid deposit in a patient. Such methodsentail administering an effective dosage of an antibody thatspecifically binds to the amyloid deposit or a component thereof to thepatient. Such methods are particularly useful for preventing or treatingAlzheimer's disease in which case the amyloid deposit is Aβ. The methodscan be used on both asymtomatic patients and those currently showingsymptoms of disease.

The antibody used in such methods can be a human, humanized, chimeric ornonhuman antibody and can be monoclonal or polyclonal. In some methods,the antibody is prepared from a human immunized with Aβ peptide, whichhuman can be the patient to be treated with antibody.

In some methods, the antibody used binds to an epitope within residues1-28 of Aβ. In some methods the antibody binds to an epitope withinresidues 1-10, and in some methods within residues 1-5. In some methods,the antibody specifically binds to Aβ peptide without binding tofull-length amyloid precursor protein (APP).

In some methods antibody is administered at a dosage of at least 1 mg/kgbody weight antibody: In some methods, the antibody is administered inmultiple dosages over a period of at least six months. In some methods,the antibody is administered as a sustained release composition. Theantibody can be administered, for example, intraperitoneally, orally,subcutaneously, intracranially, intramuscularly, topically orintravenously.

In some methods, the antibody is administered by administering apolynucleotide encoding at least one antibody chain to the patient. Thepolynucleotide is expressed to produce the antibody chain in thepatient. Optionally, the polynucleotide encodes heavy and light chainsof the antibody. The polynucleotide is expressed to produce the heavyand light chains in the patient.

In some methods, the patient is monitored for level of administeredantibody in the blood of the patient.

In another aspect, the invention provides methods of preventing ortreating Alzheimer's disease. These methods entail administering aneffective dosage of a polypeptide comprising an active fragment of Aβthat induces an immune response to Aβ in the patient. In some methods,the fragment comprises an epitope within amino acids 1-12 of Aβ. In somemethod, the fragment comprises an epitope within amino acids 1-16 of Aβ.In some methods, the fragment comprises an epitope within amino acids13-28 of Aβ. In some methods, the fragment is free of at least the 5C-terminal amino acids in Aβ43. In some methods, the fragment comprisesup to 20 contiguous amino acids from Aβ. Fragments are typicallyadministered at greater than 10 micrograms per dose per patient.

In some methods, the fragment is administered with an adjuvant thatenhances the immune response to the Aβ peptide. The adjuvant andfragment can be administered in either order of together as acomposition. The adjuvant can be, for example, alum, MPL, QS-21 orincomplete Freund's adjuvant.

The invention further provides pharmaceutical compositions comprisingactive fragments of Aβ, such as described above, and an adjuvant.

The invention further provides methods of screening an antibody to Aβ oran active fragment of Aβ for use in treatment of Alzheimer's disease.Such methods entail administering an antibody that specifically binds toAβ or a fragment of Aβ to a transgenic animal disposed to developcharacteristics of Alzheimer's disease. One then detects a reduction inthe extent or rate of development of the characteristics relative to acontrol transgenic animal as a measure of the efficacy of the antibodyor fragment. Optionally, antibodies can also be screened for capacity tobind an epitope within amino acids 1-28 or other epitope of Aβ.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Antibody titer after injection of transgenic mice with Aβ1-42.

FIG. 2: Amyloid burden in the hippocampus. The percentage of the area ofthe hippocampal region occupied by amyloid plaques, defined byreactivity with the Aβ-specific monoclonal antibody 3D6, was determinedby computer-assisted quantitative image analysis of immunoreacted brainsections. The values for individual mice are shown sorted by treatmentgroup. The horizontal line for each grouping indicates the median valueof the distribition.

FIG. 3: Neuritic dystrophy in the hippocampus. The percentage of thearea of the hippocampal region occupied by dystrophic neurites, definedby their reactivity with the human APP-specific monoclonal 8E5, wasdetermined by quantitative computer-assisted image analysis ofimmunoreacted brain sections. The values for individual mice are shownfor the AN1792-treated group and the PBS-treated control group. Thehorizontal line for each grouping indicates the median value of thedistribution.

FIG. 4: Astrocytosis in the retrosplenial cortex. The percentage of thearea of the cortical region occupied by glial fibrillary acidic protein(GFAP)-positive astrocytes was determined by quantitativecomputer-assisted image analysis of immunoreacted brain sections. Thevalues for individual mice are shown sorted by treatment group andmedian group values are indicated by horizontal lines.

FIG. 5: Geometric mean antibody titers to Aβ1-42 following immunizationwith a range of eight doses of AN1792 containing 0.14, 0.4, 1.2, 3.7,11, 33, 100, or 300 μg.

FIG. 6: Kinetics of antibody response to AN1792 immunization. Titers areexpressed as geometric means of values for the 6 animals in each group.

FIG. 7: Quantitative image analysis of the cortical amyloid burden inPBS- and AN1792-treated mice.

FIG. 8: Quantitative image analysis of the neuritic plaque burden inPBS- and AN1792-treated mice.

FIG. 9: Quantitative image analysis of the percent of the retrosplenialcortex occupied by astrocytosis in PBS- and AN1792-treated mice.

FIG. 10: Lymphocyte Proliferation Assay on spleen cells fromAN1792-treated (FIG. 10A) or PBS-treated (FIG. 10B).

FIG. 11: Total Aβ levels in the cortex. A scatterplot of individual Aβprofiles in mice immunized with Aβ or APP derivatives combined withFreund' adjuvant.

FIG. 12: Amyloid burden in the cortex was determined by quantitativeimage analysis of immunoreacted brain sections for mice immunized withthe Aβ peptide conjugates Aβ1-5, Aβ1-12, and Aβ13-28; the full length Aβaggregates AN1792 (Aβ1-42) and AN1528 (Aβ1-40) and the PBS-treatedcontrol group.

FIG. 13: Geometric mean titers of Aβ-specific antibody for groups ofmice immunized with Aβ or APP derivatives combined with Freund'sadjuvant.

FIG. 14: Geometric mean titers of Aβ-specific antibody for groups ofguinea pigs immunized with AN1792, or a palmitoylated derivativethereof, combined with various adjuvants.

FIGS. 15A-E: Aβ levels in the cortex of 12-month old PDAPP mice treatedwith AN1792 or AN1528 in combination with different adjuvants. The Aβlevel for individual mice in each treatment group, and the median, mean,and p values for each treatment group are shown.

FIG. 15A: The values for mice for the PBS-treated control group and theuntreated control group.

FIG. 15B: The values for mice in the AN1528/alum andAN1528/MPL-treatment groups.

FIG. 15C: The values for mice in the AN1528/QS21 and AN1792/Freund'sadjuvant treatment groups.

FIG. 15D: The values for mice in the AN1792/Thimerosol and AN1792/alumtreatment groups.

FIG. 15E: The values for mice in the AN1792/MPL and AN1792/QS21treatment groups.

FIG. 16: Mean titer of mice treated with polyclonal antibody to Aβ.

FIG. 17: Mean titer of mice treated with monoclonal antibody 10D5 to Aβ.

FIG. 18: Mean titer of mice treated with monoclonal antibody 2F12 to Aβ.

DEFINITIONS

The term “substantial identity” means that two peptide sequences, whenoptimally aligned, such as by the programs GAP or BESTFIT using defaultgap weights, share at least 65 percent sequence identity, preferably atleast 80 or 90 percent sequence identity, more preferably at least 95percent sequence identity or more (e.g., 99 percent sequence identity orhigher). Preferably, residue positions which are not identical differ byconservative amino acid substitutions.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, J.Mol. Biol 48:443 (1970), by the search for similarity method of Pearson& Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by visual inspection (see generallyAusubel et al., supra). One example of algorithm that is suitable fordetermining percent sequence identity and sequence similarity is theBLAST algorithm, which is described in Altschul et al, J. Mol. Biol.215:403-410 (1990). Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Information(http://www.ncbi.nlm.nih.gov/). Typically, default program parameterscan be used to perform the sequence comparison, although customizedparameters can also be used. For amino acid sequences, the BLASTPprogram uses as defaults a wordlength (W) of 3, an expectation (E) of10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc.Natl. Acad. Sci. USA 89, 10915 (1989))

For purposes of classifying amino acids substitutions as conservative ornonconservative, amino acids are grouped as follows: Group I(hydrophobic sidechains): norleucine, met, ala, val, leu, ile; Group II(neutral hydrophilic side chains): cys, ser, thr; Group III (acidic sidechains): asp, glu; Group IV (basic side chains): asn, gin, his, lys,arg; Group V (residues influencing chain orientation): gly, pro; andGroup VI (aromatic side chains): trp, tyr, phe. Conservativesubstitutions involve substitutions between amino acids in the sameclass. Non-conservative substitutions constitute exchanging a member ofone of these classes for a member of another.

Therapeutic agents of the invention are typically substantially purefrom undesired contaminant. This means that an agent is typically atleast about 50% w/w (weight/weight) purity, as well as beingsubstantially free from interfering proteins and contaminants. Sometimesthe agents are at least about 80% w/w and, more preferably at least 90or about 95% w/w purity. However, using conventional proteinpurification techniques, homogeneous peptides of at least 99% w/w can beobtained.

Specific binding between two entities means an affinity of at least 10⁶,10⁷, 10^(8,) 10⁹ M⁻¹, or 10¹⁰ M⁻¹. Affinities greater than 10⁸ M⁻¹ arepreferred.

The term “antibody” or “immunoglobulin” is used to include intactantibodies and binding fragments thereof. Typically, fragments competewith the intact antibody from which they were derived for specificbinding to an antigen fragments including separate heavy chains, lightchains Fab, Fab′ F(ab′)2, Fabc, and Fv. Fragments are produced byrecombinant DNA techniques, or by enzymatic or chemical separation ofintact immunoglobulins. The term “antibody” also includes one or moreimmunoglobulin chain that are chemically conjugated to, or expressed as,fusion proteins with other proteins. The term “antibody” also includesbispecific antibody. A bispecific or bifunctional antibody is anartificial hybrid antibody having two different heavy/light chain pairsand two different binding sites. Bispecific antibodies can be producedby a variety of methods including fusion of hybridomas or linking ofFab′ fragments. See, e.g., Songsivilai & Lachmann, Clin. Exp. Immunol.79:315-321 (1990); Kostelny et al., J. Immunol. 148, 1547-1553 (1992).

APP⁶⁹⁵, APP⁷⁵¹, and APP⁷⁷⁰ refer, respectively, to the 695, 751, and 770amino acid residue long polypeptides encoded by the human APP gene. SeeKang et al., Nature 325, 773 (1987); Ponte et al., Nature 331, 525(1988); and Kitaguchi et al., Nature 331, 530 (1988). Amino acids withinthe human amyloid precursor protein (APP) are assigned numbers accordingto the sequence of the APP770 isoform. Terms such as Aβ39, Aβ40, Aβ41,Aβ42 and Aβ43 refer to an Aβ peptide containing amino acid residues1-39, 1-40, 1-41, 1-42 and 1-43.

The term “epitope” or “antigenic determinant” refers to a site on anantigen to which B and/or T cells respond. B-cell epitopes can be formedboth from contiguous amino acids or noncontiguous amino acids juxtaposedby tertiary folding of a protein. Epitopes formed from contiguous aminoacids are typically retained on exposure to denaturing solvents whereasepitopes formed by tertiary folding are typically lost on treatment withdenaturing solvents. An epitope typically includes at least 3, and moreusually, at least 5 or 8-10 amino acids in a unique spatialconformation. Methods of determining spatial conformation of epitopesinclude, for example, x-ray crystallography and 2-dimensional nuclearmagnetic resonance. See, e.g., Epitope Mapping Protocols in Methods inMolecular Biology, Vol. 66, Glenn E. Morris, Ed. (1996). Antibodies thatrecognize the same epitope can be identified in a simple immunoassayshowing the ability of one antibody to block the binding of anotherantibody to a target antigen. T-cells recognize continuous epitopes ofabout nine amino acids for CD8 cells or about 13-15 amino acids for CD4cells. T cells that recognize the epitope can be identified by in vitroassays that measure antigen-dependent proliferation, as determined by³H-thymidine incorporation by primed T cells in response to an epitope(Burke et al., J. Inf. Dis. 170, 1110-19 (1994)), by antigen-dependentkilling (cytotoxic T lymphocyte assay, Tigges et al., J. Immunol. 156,3901-3910) or by cytokine secretion.

The term “immunological” or “immune” response is the development of abeneficial humoral (antibody mediated) and/or a cellular (mediated byantigen-specific T cells or their secretion products) response directedagainst an amyloid peptide in a recipient patient. Such a response canbe an active response induced by administration of immunogen or apassive response induced by administration of antibody or primedT-cells. A cellular immune response is elicited by the presentation ofpolypeptide epitopes in association with Class I or Class II MHCmolecules to activate antigen-specific CD4⁺ T helper cells and/or CD8⁺cytotoxic T cells. The response may also involve activation ofmonocytes, macrophages, NK cells, basophils, dendritic cells,astrocytes, microglia cells, eosinophils or other components of innateimmunity. The presence of a cell-mediated immunological response can bedetermined by proliferation assays (CD4⁺ T cells) or CTL (cytotoxic Tlymphocyte) assays (see Burke, supra; Tigges, supra). The relativecontributions of humoral and cellular responses to the protective ortherapeutic effect of an immunogen can be distinguished by separatelyisolating antibodies and T-cells from an immunized syngeneic animal andmeasuring protective or therapeutic effect in a second subject.

An “immunogenic agent” or “immunogen” is capable of inducing animmunological response against itself on administration to a patient,optionally in conjunction with an adjuvant.

The term “naked polynucleotide” refers to a polynucleotide not complexedwith colloidal materials. Naked polynucleotides are sometimes cloned ina plasmid vector.

The term “adjuvant” refers to a compound that when administered inconjunction with an antigen augments the immune response to the antigen,but when administered alone does not generate an immune response to theantigen. Adjuvants can augment an immune response by several mechanismsincluding lymphocyte recruitment, stimulation of B and/or T cells, andstimulation of macrophages.

The term “patient” includes human and other mammalian subjects thatreceive either prophylactic or therapeutic treatment.

Disaggregated or monomeric Aβ means soluble, monomeric peptide units ofAβ. One method to prepare monomeric Aβ is to dissolve lyophilizedpeptide in neat DMSO with sonication. The resulting solution iscentrifuged to remove any insoluble particulates. Aggregated Aβ is amixture of oligomers in which the monomeric units are held together bynoncovalent bonds.

Competition between antibodies is determined by an assay in which theimmunoglobulin under test inhibits specific binding of a referenceantibody to a common antigen, such as Aβ. Numerous types of competitivebinding assays are known, for example: solid phase direct or indirectradioimmunoassay (RIA), solid phase direct or indirect enzymeimmunoassay (EIA), sandwich competition assay (see Stahli et al.,Methods in Enzymology 9:242-253 (1983)); solid phase directbiotin-avidin EIA (see Kirkland et al., J. Immunol. 137:3614-3619(1986)); solid phase direct labeled assay, solid phase direct labeledsandwich assay (see Harlow and Lane, “Antibodies, A Laboratory Manual,”Cold Spring Harbor Press (1988)); solid phase direct label RIA usingI-125 label (see Morel et al., Molec. Immunol. 25(1):7-15 (1988)); solidphase direct biotin-avidin EIA (Cheung et al., Virology 176:546-552(1990)); and direct labeled RIA (Moldenhauer et al., Scand. J. Immunol.32:77-82 (1990)). Typically, such an assay involves the use of purifiedantigen bound to a solid surface or cells bearing either of these, anunlabelled test immunoglobulin and a labelled reference immunoglobulin.Competitive inhibition is measured by determining the amount of labelbound to the solid surface or cells in the presence of the testimmunoglobulin. Usually the test immunoglobulin is present in excess.Antibodies identified by competition assay (competing antibodies)include antibodies binding to the same epitope as the reference antibodyand antibodies binding to an adjacent epitope sufficiently proximal tothe epitope bound by the reference antibody for steric hindrance tooccur. Usually, when a competing antibody is present in excess, it willinhibit specific binding of a reference antibody to a common antigen byat least 50 or 75%.

Compositions or methods “comprising” one or more recited elements mayinclude other elements not specifically recited. For example, acomposition that comprises Aβ peptide encompasses both an isolated Aβpeptide and Aβ peptide as a component of a larger polypeptide sequence.

DETAILED DESCRIPTION

I. General

The invention provides pharmaceutical compositions and methods forprophylactic and therapeutic treatment of diseases characterized byaccumulation of amyloid deposits. Amyloid deposits comprise a peptideaggregated to an insoluble mass. The nature of the peptide varies indifferent diseases but in most cases, the aggregate has a β-pleatedsheet structure and stains with Congo Red dye. Diseases characterized byamyloid deposits include Alzheimer's disease (AD), both late and earlyonset. In both diseases, the amyloid deposit comprises a peptide termedAβ, which accumulates in the brain of affected individuals. Examples ofsome other diseases characterized by amyloid deposits are SAAamyloidosis, hereditary Icelandic syndrome, multiple myeloma, andspongiform encephalopathies, including mad cow disease, CreutzfeldtJakob disease, sheep scrapie, and mink spongiform encephalopathy (seeWeissmann et al., Curr. Opin. Neurobiol. 7, 695-700 (1997); Smits etal., Veterinary Quarterly 19, 101-105 (1997); Nathanson et al., Am. J.Epidemiol. 145, 959-969 (1997)). The peptides forming the aggregates inthese diseases are serum amyloid A, cystantin C, IgG kappa light chainrespectively for the first three, and prion protein for the others.

II. Therapeutic Agents

A. Alzheimer's Disease

1. Agents Inducing Active Immune Response

Therapeutic agents for use in the present invention induce an immuneresponse against Aβ peptide. These agents include Aβ peptide itself andvariants thereof, analogs and mimetics of Aβ peptide that induce and/orcrossreact with antibodies to Aβ peptide, and antibodies or T-cellsreactive with Aβ peptide. Induction of an immune response can be activeas when an immunogen is administered to induce antibodies or T-cellsreactive with Aβ in a patient, or passive, as when an antibody isadministered that itself binds to Aβ in patient.

Aβ, also known as β-amyloid peptide, or A4 peptide (see U.S. Pat. No.4,666,829; Glenner & Wong, Biochem. Biophys. Res. Commun. 120, 1131(1984)), is a peptide of 39-43 amino acids, which is the principalcomponent of characteristic plaques of Alzheimer's disease. Aβ isgenerated by processing of a larger protein APP by two enzymes, termed βand γ secretases (see Hardy, TINS 20, 154 (1997)). Known mutations inAPP associated with Alzheimer's disease occur proximate to the site of βor γ secretase, or within Aβ. For example, position 717 is proximate tothe site of γ-secretase cleavage of APP in its processing to Aβ, andpositions 670/671 are proximate to the site of β-secretase cleavage. Itis believed that the mutations cause AD by interacting with the cleavagereactions by which Aβ is formed so as to increase the amount of the42/43 amino acid form of Aβ generated.

Aβ has the unusual property that it can fix and activate both classicaland alternate complement cascades. In particular, it binds to Clq andultimately to C3bi. This association facilitates binding to macrophagesleading to activation of B cells. In addition, C3bi breaks down furtherand then binds to CR2 on B cells in a T cell dependent manner leading toa 10,000 increase in activation of these cells. This mechanism causes Aβto generate an immune response in excess of that of other antigens.

The therapeutic agent used in the claimed methods can be any of thenaturally occurring forms of Aβ peptide, and particularly the humanforms (i.e., Aβ39, Aβ40, Aβ41, Aβ42 or Aβ43). The sequences of thesepeptides and their relationship to the APP precursor are illustrated byFIG. 1 of Hardy et al., TINS 20, 155-158 (1997). For example, Aβ42 hasthe sequence: (SEQ ID NO:1)H₂N-Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln-Lys-Leu-Val-Phe-Phe-Ala-Glu-Asp-Val-Gly-Ser-Asn-Lys-Gly-Ala-Ile-Ile-GIy-Leu-Met-Val-Gly-Gly-Val-Val-IIe-Ala-OH.

Aβ41, Aβ40 and Aβ39 differ from Aβ42 by the omission of Ala, Ala-Ile,and Ala-Ile-Val respectively from the C-terminal end. Aβ43 differs fromAβ42 by the presence of a threonine residue at the C-terminus. Thetherapeutic agent can also be an active fragment or analog of a naturalAβ peptide that contains an epitope that induces a similar protective ortherapeutic immune response on administration to a human. Immunogenicfragments typically have a sequence of at least 3, 5, 6, 10 or 20contiguous amino acids from a natural peptide. Immunogenic fragmentsinclude Aβ1-5, 1-6, 1-12, 13-28, 17-28, 1-28, 25-35, 35-40 and 35-42.Fragments lacking at least one, and sometimes at least 5 or 10C-terminal amino acid present in a naturally occurring forms of Aβ areused in some methods. For example, a fragment lacking 5 amino acids fromthe C-terminal end of AB43 includes the first 38 amino acids from theN-terminal end of AB. Fragments from the N-terminal half of Aβ arepreferred in some methods. Analogs include allelic, species and inducedvariants. Analogs typically differ from naturally occurring peptides atone or a few positions, often by virtue of conservative substitutions.Analogs typically exhibit at least 80 or 90% sequence identity withnatural peptides. Some analogs also include unnatural amino acids ormodifications of N or C terminal amino acids. Examples of unnaturalamino acids are α, α-disubstituted amino acids, N-alkyl amino acids,lactic acid, 4-hydroxyproline, γ-carboxyglutamate,γ-N,N,N-trimethyllysine, γ-N-acetyllysine, O-phosphoserine,N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine,ω-N-methylarginine. Fragments and analogs can be screened forprophylactic or therapeutic efficacy in transgenic animal models incomparison with untreated or placebo controls as described below.

Aβ, its fragments, analogs and other amyloidogenic peptides can besynthesized by solid phase peptide synthesis or recombinant expression,or can be obtained from natural sources; Automatic peptide synthesizersare commercially available from numerous suppliers, such as AppliedBiosystems, Foster City, Calif. Recombinant expression can be inbacteria, such as E. coli, yeast, insect cells or mammalian cells.Procedures for recombinant expression are described by Sambrook et al.,Molecular Cloning: A Laboratory Manual (C.S.H.P. Press, NY 2d ed.,1989). Some forms of Aβ peptide are also available commercially (e.g.,American Peptides Company, Inc., Sunnyvale, Calif. and CaliforniaPeptide Research, Inc. Napa, Calif.).

Therapeutic agents also include longer polypeptides that include, forexample, an Aβ peptide, active fragment or analog together with otheramino acids. For example, Aβ peptide can be present as intact APPprotein or a segment thereof, such as the C-100 fragment that begins atthe N-terminus of Aβ and continues to the end of APP. Such polypeptidescan be screened for prophylactic or therapeutic efficacy in animalmodels in comparison with untreated or placebo controls as describedbelow. The Aβ peptide, analog, active fragment or other polypeptide canbe administered in associated form (i.e., as an β-amyloid peptide) or indissociated form. Therapeutic agents also include multimers of monomericimmunogenic agents.

In a further variation, an immunogenic peptide, such as Aβ, can bepresented as a viral or bacterial vaccine. A nucleic acid encoding theimmunogenic peptide is incorporated into a genome or episome of thevirus or bacteria. Optionally, the nucleic acid is incorporated in sucha manner that the immunogenic peptide is expressed as a secreted proteinor as a fusion protein with an outersurface protein of a virus or atransmembrane protein of a bacteria so that the peptide is displayed.Viruses or bacteria used in such methods should be nonpathogenic orattenuated. Suitable viruses include adenovirus, HSV, vaccinia and fowlpox. Fusion of an immunogenic peptide to HBsAg of HBV is particularlysuitable. Therapeutic agents also include peptides and other compoundsthat do not necessarily have a significant amino acid sequencesimilarity with Aβ but nevertheless serve as mimetics of Aβ and induce asimilar immune response. For example, any peptides and proteins formingβ-pleated sheets can be screened for suitability. Anti-idiotypicantibodies against monoclonal antibodies to Aβ or other amyloidogenicpeptides can also be used. Such anti-Id antibodies mimic the antigen andgenerate an immune response to it (see Essential Immunology (Roit ed.,Blackwell Scientific Publications, Palo Alto, 6th ed.), p. 181).

Random libraries of peptides or other compounds can also be screened forsuitability. Combinatorial libraries can be produced for many types ofcompounds that can be synthesized in a step-by-step fashion. Suchcompounds include polypeptides, beta-turn mimetics, polysaccharides,phospholipids, hormones, prostaglandins, steroids, aromatic compounds,heterocyclic compounds, benzodiazepines, oligomeric N-substitutedglycines and oligocarbamates. Large combinatorial libraries of thecompounds can be constructed by the encoded synthetic libraries (ESL)method described in Affymax, WO 95/12608, Affymax, WO 93/06121, ColumbiaUniversity, WO 94/08051, Pharmacopeia, WO 95/35503 and Scripps, WO95/30642 (each of which is incorporated by reference for all purposes).Peptide libraries can also be generated by phage display methods. See,e.g., Devlin, WO 91/18980.

Combinatorial libraries and other compounds are initially screened forsuitability by determining their capacity to bind to antibodies orlymphocytes (B or T) known to be specific for Aβ or other amyloidogenicpeptides. For example, initial screens can be performed with anypolyclonal sera or monoclonal antibody to Aβ or other amyloidogenicpeptide. Compounds identified by such screens are then further analyzedfor capacity to induce antibodies or reactive lymphocytes to Aβ or otheramyloidogenic peptide. For example, multiple dilutions of sera can betested on microtiter plates that have been precoated with Aβ peptide anda standard ELISA can be performed to test for reactive antibodies to Aβ.Compounds can then be tested for prophylactic and therapeutic efficacyin transgenic animals predisposed to an amyloidogenic disease, asdescribed in the Examples. Such animals include, for example, micebearing a 717 mutation of APP described by Games et al., supra, and micebearing a Swedish mutation of APP such as described by McConlogue etal., U.S. Pat. No. 5,612,486 and Hsiao et al., Science 274, 99 (1996);Staufenbiel et al., Proc. Natl. Acad. Sci. USA 94, 13287-13292 (1997);Sturchler-Pierrat et al., Proc. Natl. Acad. Sci. USA 94, 13287-13292(1997); Borchelt et al., Neuron 19, 939-945 (1997)). The same screeningapproach can be used on other potential agents such as fragments of Aβ,analogs of Aβ and longer peptides including Aβ, described above.

2. Agents Inducing Passive Immune Response

Therapeutic agents of the invention also include antibodies thatspecifically bind to Aβ or other component of amyloid plaques. Suchantibodies can be monoclonal or polyclonal. Some such antibodies bindspecifically to the aggregated form of Aβ without binding to thedissociated form. Some bind specifically to the dissociated form withoutbinding to the aggregated form. Some bind to both aggregated anddissociated forms. Some such antibodies bind to a naturally occurringshort form of Aβ (i.e., Aβ39, 40 or 41) without binding to a naturallyoccurring long form of Aβ (i.e., Aβ42 and Aβ43). Some antibodies bind toa long form without binding to a short form. Some antibodies bind to Aβwithout binding to full-length amyloid precursor protein. Someantibodies bind to Aβ with a binding affinity greater than or equal toabout 10⁶, 10⁷, 10⁸, 10⁹, or 10¹⁰ M⁻¹.

Polyclonal sera typically contain mixed populations of antibodiesbinding to several epitopes along the length of Aβ. Monoclonalantibodies bind to a specific epitope within Aβ that can be aconformational or nonconformational epitope. Some monoclonal antibodiesbind to an epitope within residues 1-28 of Aβ (with the first N terminalresidue of natural Aβ designated 1). Some monoclonal antibodies bind toan epitope within residues 1-10 of Aβ Some monoclonal antibodies bind toan epitope within residues 1-16 of Aβ. Some monoclonal antibodies bindto an epitope within residues 1-25 of Aβ. Some monoclonal antibodiesbind to an epitope within amino acids 1-5, 5-10, 10-15, 15-20, 25-30,10-20, 20-30, or 10-25 of Aβ. Prophylactic and therapeutic efficacy ofantibodies can be tested using the transgenic animal model proceduresdescribed in the Examples.

i. General Characteristics of Immunoglobulins

The basic antibody structural unit is known to comprise a tetramer ofsubunits. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kDa) and one“heavy” chain (about 50-70 kDa). The amino-terminal portion of eachchain includes a variable region of about 100 to 110 or more amino acidsprimarily responsible for antigen recognition. The carboxy-terminalportion of each chain defines a constant region primarily responsiblefor effector function.

Light chains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, and define theantibody's isotype as IgG, IgM, IgA, IgD and IgE, respectively. Withinlight and heavy chains, the variable and constant regions are joined bya “J” region of about 12 or more amino acids, with the heavy chain alsoincluding a “D” ion of about 10 more amino acids. (See generally,Fundamental Immunology (Paul, W., ed., 2nd ed. Raven Press, N.Y., 1989),Ch. 7 (incorporated by reference in its entirety for all purposes).

The variable regions of each light/heavy chain pair form the antibodybinding site. Thus, and intact antibody has two binding sites. Except inbifunctional or bispecific antibodies, the two binding sites are thesame. The chains all exhibit the same general structure of relativelyconserved framework regions (FR) joined by three hypervariable regions,also called complementarity determining regions or CDRs. The CDRs fromthe two chains of each pair are aligned by the framework regions,enabling binding to a specific epitope. From N-terminal to C-terminal,both light and heavy chains comprise the domains FR1, CDR1, FR2, CDR2,FR3, CDR3 and FR4. The assignment of amino acids to each domain is inaccordance with the definitions of Kabat, Sequences of Proteins ofImmunological Interest (National Institutes of Health, Bethesda, Md.,1987 and 1991), or Chothia & Lesk, J. Mol. Biol. 196:901-917 (1987);Chothia et al., Nature 342:878-883 (1989).

ii. Production of Nonhuman Antibodies

The production of non-human monoclonal antibodies, e.g., murine, guineapig, rabbit or rat, can be accomplished by, for example, immunizing theanimal with Aβ. A longer polypeptide comprising Aβ or an immunogenicfragment of Aβ or anti-idiotypic antibodies to an antibody to Aβ. SeeHarlow & Lane, Antibodies, A Laboratory Manual (CSHP NY, 1988)(incorporated by reference for all purposes). Such an immunogen can beobtained from a natural source, by peptide synthesis or by recombinantexpression. Optionally, the immunogen can be administered fused orotherwise complexed with a carrier protein, as described below.Optionally, the immunogen can be administered with an adjuvant. Severaltypes of adjuvant can be used as described below. Complete Freund'sadjuvant followed by incomplete adjuvant is preferred for immunizationof laboratory animals. Rabbits or guinea pigs are typically used formaking polyclonal antibodies. Mice are typically used for makingmonoclonal antibodies. Antibodies are screened for specific binding toAβ. Optionally, antibodies are further screened for binding to aspecific region of Aβ. The latter screening can be accomplished bydetermining binding of an antibody to a collection of deletion mutantsof an Aβ peptide and determining which deletion mutants bind to theantibody. Binding can be assessed, for example by Western blot or ELISA.The smallest fragment to show specific binding to the antibody definesthe epitope of the antibody. Alternatively, epitope specificity can bedetermined by a competition assay is which a test and reference antibodycompete for binding to Aβ. If the test and reference antibodies compete,then they bind to the same epitope or epitopes sufficiently proximalthat binding of one antibody interferes with binding of the other.

iii. Chimeric and Humanized Antibodies

Chimeric and humanized antibodies have the same or similar bindingspecificity and affinity as a mouse or other nonhuman antibody thatprovides the starting material for construction of a chimeric orhumanized antibody. Chimeric antibodies are antibodies whose light andheavy chain genes have been constructed, typically by geneticengineering, from immunoglobulin gene segments belonging to differentspecies. For example, the variable (V) segments of the genes from amouse monoclonal antibody may be joined to human constant (C) segments,such as IgG1 and IgG4. A typical chimeric antibody is thus a hybridprotein consisting of the V or antigen-binding domain from a mouseantibody and the C or effector domain from a human antibody.

Humanized antibodies have variable region framework residuessubstantially from a human antibody (termed an acceptor antibody) andcomplementarity determining regions substantially from a mouse-antibody,(referred to as the donor immunoglobulin). See, Queen et al., Proc.Natl. Acad. Sci. USA 86:10029-10033 (1989) and WO 90/07861, U.S. Pat.No. 5,693,762, U.S. Pat. No. 5,693,761, U.S. Pat. No. 5,585,089, U.S.Pat. No. 5,530,101 and Winter, U.S. Pat. No. 5,225,539 (incorporated byreference in their entirety for all purposes). The constant region(s),if present, are also substantially or entirely from a humanimmunoglobulin. The human variable domains are usually chosen from humanantibodies whose framework sequences exhibit a high degree of sequenceidentity with the murine variable region domains from which the CDRswere derived. The heavy and light chain variable region frameworkresidues can be derived from the same or different human antibodysequences. The human antibody sequences can be the sequences ofnaturally occurring human antibodies or can be consensus sequences ofseveral human antibodies. See Carter et al., WO 92/22653. Certain aminoacids from the human variable region framework residues are selected forsubstitution based on their possible influence on CDR conformationand/or binding to antigen. Investigation of such possible influences isby modeling, examination of the characteristics of the amino acids atparticular locations, or empirical observation of the effects ofsubstitution or mutagenesis of particular amino acids.

For example, when an amino acid differs between a murine variable regionframework residue and a selected human variable region frameworkresidue, the human framework amino acid should usually be substituted bythe equivalent framework amino acid from the mouse antibody when it isreasonably expected that the amino acid:

-   -   (1) noncovalently binds antigen directly,    -   (2) is adjacent to a CDR region,    -   (3) otherwise interacts with a CDR region (e.g. is within about        6 A of a CDR region), or    -   (4) participates in the VL-VH interface.

Other candidates for substitution are acceptor human framework aminoacids that are unusual for a human immunoglobulin at that position.These amino acids can be substituted with amino acids from theequivalent position of the mouse donor antibody or from the equivalentpositions of more typical human immunoglobulins. Other candidates forsubstitution are acceptor human framework amino acids that are unusualfor a human immunoglobulin at that position. The variable regionframeworks of humanized immunoglobulins usually show at least 85%sequence identity to a human variable region framework sequence orconsensus of such sequences.

iv. Human Antibodies

Human antibodies against Aβ are provided by a variety of techniquesdescribed below. Some human antibodies are selected by competitivebinding experiments, or otherwise, to have the same epitope specificityas a particular mouse antibody, such as one of the mouse monoclonalsdescribed in Example XI. Human antibodies can also be screened for aparticular epitope specificity by using only a fragment of Aβ as theimmunogen, and/or by screening antibodies against a collection ofdeletion mutants of Aβ.

(1) Trioma Methodology

The basic approach and an exemplary cell fusion partner, SPAZ-4, for usein this approach have been described by Oestberg et al., Hybridoma2:361-367 (1983); Oestberg, U.S. Pat. No.4,634,664; and Engleman et al.,U.S. Pat. No. 4,634,666 (each of which is incorporated by reference inits entirety for all purposes). The antibody-producing cell linesobtained by this method are called triomas, because they are descendedfrom three cells—two human and one mouse. Initially, a mouse myelomaline is fused with a human B-lymphocyte to obtain anon-antibody-producing xenogeneic hybrid cell, such as the SPAZ-4 cellline described by Oestberg, supra. The xenogeneic cell is then fusedwith an immunized human B-lymphocyte to obtain an antibody-producingtrioma cell line. Triomas have been found to produce antibody morestably than ordinary hybridomas made from human cells.

The immunized B-lymphocytes are obtained from the blood, spleen, lymphnodes or bone marrow of a human donor. If antibodies against a specificantigen or epitope are desired, it is preferable to use that antigen orepitope thereof for immunization. Immunization can be either in vivo orin vitro. For in vivo immunization, B cells are typically isolated froma human immunized with Aβ, a fragment thereof, larger polypeptidecontaining Aβ or fragment, or an anti-idiotypic antibody to an antibodyto Aβ. In some methods, B cells are isolated from the same patient whois ultimately to be administered antibody therapy. For in vitroimmunization, B-lymphocytes are typically exposed to antigen for aperiod of 7-14 days in a media such as RPMI-1640 (see Engleman, supra)supplemented with 10% human plasma.

The immunized B-lymphocytes are fused to a xenogeneic hybrid cell suchas SPAZ-4 by well known methods. For example, the cells are treated with40-50% polyethylene glycol of MW 1000-4000, at about 37 degrees, forabout 5-10 min. Cells are separated from the fusion mixture andpropagated in media selective for the desired hybrids (e.g., HAT or AH).Clones secreting antibodies having the required binding specificity areidentified by assaying the trioma culture medium for the ability to bindto Aβ or a fragment thereof Triomas producing human antibodies havingthe desired specificity are subcloned by the limiting dilution techniqueand grown in vitro in culture medium. The trioma cell lines obtained arethen tested for the ability to bind Aβ or a fragment thereof.

Although triomas are genetically stable they do not produce antibodiesat very high levels. Expression levels can be increased by cloningantibody genes from the trioma into one or more expression vectors, andtransforming the vector into standard mammalian, bacterial or yeast celllines.

(2) Transgenic Non-Human Mammals

Human antibodies against Aβ can also be produced from non-humantransgenic mammals having transgenes encoding at least a segment of thehuman immunoglobulin locus. Usually, the endogenous immunoglobulin locusof such transgenic mammals is functionally inactivated. Preferably, thesegment of the human immunoglobulin locus includes unrearrangedsequences of heavy and light chain components. Both inactivation ofendogenous immunoglobulin genes and introduction of exogenousimmunoglobulin genes can be achieved by targeted homologousrecombination, or by introduction of YAC chromosomes. The transgenicmammals resulting from this process are capable of functionallyrearranging the immunoglobulin component sequences, and expressing arepertoire of antibodies of various isotypes encoded by humanimmunoglobulin genes, without expressing endogenous immunoglobulingenes. The production and properties of mammals having these propertiesare described in detail by, e.g., Lonberg et al., WO93/12227 (1993);U.S. Pat. No. 5,877,397, U.S. Pat. No. 5,874,299, U.S. Pat. No.5,814,318, U.S. Pat. No. 5,789,650, U.S. Pat. No. 5,770,429, U.S. Pat.No. 5,661,016, U.S. Pat. No. 5,633,425, U.S. Pat. No. 5,625,126, U.S.Pat. No. 5,569,825, U.S. Pat. No. 5,545,806, Nature 148, 1547-1553(1994), Nature Biotechnology 14, 826 (1996), Kucherlapati, WO 91/10741(1991) (each of which is incorporated by reference in its entirety forall purposes). Transgenic mice are particularly suitable. Anti-Aβantibodies are obtained by immunizing a transgenic nonhuman mammal, suchas described by Lonberg or Kucherlapati, supra, with Aβ or a fragmentthereof. Monoclonal antibodies are prepared by, e.g., fusing B-cellsfrom such mammals to suitable myeloma cell lines using conventionalKohler-Milstein technology. Human polyclonal antibodies can also beprovided in the form of serum from humans immunized with an immunogenicagent. Optionally, such polyclonal antibodies can be concentrated byaffinity purification using Aβ or other amyloid peptide as an affinityreagent.

(3) Phage Display Methods

A further approach for obtaining human anti-Aβ antibodies is to screen aDNA library from human B cells according to the general protocoloutlined by Huse et al., Science 246:1275-1281 (1989). As described fortrioma methodology, such B cells can be obtained from a human immunizedwith Aβ, fragments, longer polypeptides containing Aβ or fragments oranti-idiotypic antibodies. Optionally, such B cells are obtained from apatient who is ultimately to receive antibody treatment. Antibodiesbinding to Aβ or a fragment thereof are selected. Sequences encodingsuch antibodies (or a binding fragments) are then cloned and amplified.The protocol described by Huse is rendered more efficient in combinationwith phage-display technology. See, e.g., Dower et al., WO 91/17271 andMcCafferty et al., WO 92/01047, U.S. Pat. No. 5,877,218, U.S. Pat. No.5,871,907, U.S. Pat. No. 5,858,657, U.S. Pat. No. 5,837,242, U.S. Pat.No. 5,733,743 and U.S. Pat. No. 5,565,332 (each of which is incorporatedby reference in its entirety for all purposes). In these methods,libraries of phage are produced in which members display differentantibodies on their outer surfaces. Antibodies are usually displayed asFv or Fab fragments. Phage displaying antibodies with a desiredspecificity are selected by affinity enrichment to an Aβ peptide orfragment thereof.

In a variation of the phage-display method, human antibodies having thebinding specificity of a selected murine antibody can be produced. SeeWinter, WO 92/20791. In this method, either the heavy or light chainvariable region of the selected murine antibody is used as a startingmaterial. If, for example, a light chain variable region is selected asthe starting material, a phage library is constructed in which membersdisplay the same light chain variable region (i.e., the murine startingmaterial) and a different heavy chain variable region. The heavy chainvariable regions are obtained from a library of rearranged human heavychain variable regions. A phage showing strong specific binding for Aβ(e.g., at least 10⁸ and preferably at least 10⁹ M⁻¹) is selected. Thehuman heavy chain variable region from this phage then serves as astarting material for constructing a further phage library. In thislibrary, each phage displays the same heavy chain variable region (i.e.,the region identified from the first display library) and a differentlight chain variable region. The light chain variable regions areobtained from a library of rearranged human variable light chainregions. Again, phage showing strong specific binding for Aβareselected. These phage display the variable regions of completely humananti-Aβ antibodies. These antibodies usually have the same or similarepitope specificity as the murine starting material.

v. Selection of Constant Region

The heavy and light chain variable regions of chimeric, humanized, orhuman antibodies can be linked to at least a portion of a human constantregion. The choice of constant region depends, in part, whetherantibody-dependent complement and/or cellular mediated toxicity isdesired. For example, isotopes IgG1 and IgG3 have complement activityand isotypes IgG2 and IgG4 do not. Choice of isotype can also affectpassage of antibody into the brain. Light chain constant regions can belambda or kappa. Antibodies can be expressed as tetramers containing twolight and two heavy chains, as separate heavy chains, light chains, asFab, Fab′ F(ab′)2, and Fv, or as single chain antibodies in which heavyand light chain variable domains are linked through a spacer.

vi. Expression of Recombinant Antibodies

Chimeric, humanized and human antibodies are typically produced byrecombinant expression. Recombinant polynucleotide constructs typicallyinclude an expression control sequence operably linked to the codingsequences of antibody chains, including naturally-associated orheterologous promoter regions. Preferably, the expression controlsequences are eukaryotic promoter systems in vectors capable oftransforming or transfecting eukaryotic host cells. Once the vector hasbeen incorporated into the appropriate host, the host is maintainedunder conditions suitable for high level expression of the nucleotidesequences, and the collection and purification of the crossreactingantibodies.

These expression vectors are typically replicable in the host organismseither as episomes or as an integral part of the host chromosomal DNA.Commonly, expression vectors contain selection markers, e.g.,ampicillin-resistance or hygromycin-resistance, to permit detection ofthose cells transformed with the desired DNA sequences.

E. coli is one prokaryotic host particularly useful for cloning the DNAsequences of the present invention. Microbes, such as yeast are alsouseful for expression. Saccharomyces is a preferred yeast host, withsuitable vectors having expression control sequences, an origin ofreplication, termination sequences and the like as desired. Typicalpromoters include 3-phosphoglycerate kinase and other glycolyticenzymes. Inducible yeast promoters include, among others, promoters fromalcohol dehydrogenase, isocytochrome C, and enzymes responsible formaltose and galactose utilization.

Mammalian cells are a preferred host for expressing nucleotide segmentsencoding immunoglobulins or fragments thereof. See Winnacker, From Genesto Clones, (VCH Publishers, NY, 1987). A number of suitable host celllines capable of secreting intact heterologous proteins have beendeveloped in the art, and include CHO cell lines, various COS celllines, HeLa cells, L cells and myeloma cell lines. Preferably, the cellsare nonhuman. Expression vectors for these cells can include expressioncontrol sequences, such as an origin of replication, a promoter, anenhancer (Queen et al., Immunol. Rev. 89:49 (1986)), and necessaryprocessing information sites, such as ribosome binding sites, RNA splicesites, polyadenylation sites, and transcriptional terminator sequences.Preferred expression control sequences are promoters derived fromendogenous genes, cytomegalovirus, SV40, adenovirus, bovinepapillomavirus, and the like. See Co et al., J. Immunol. 148:1149(1992).

Alternatively, antibody coding sequences can be incorporated intransgenes for introduction into the genome of a transgenic animal andsubsequent expression in the milk of the transgenic animal (see, e.g.,U.S. Pat. No. 5,741,957, U.S. Pat. No. 5,304,489, U.S. Pat. No.5,849,992). Suitable transgenes include coding sequences for lightand/or heavy chains in operable linkage with a promoter and enhancerfrom a mammary gland specific gene, such as casein or betalactoglobulin.

The vectors containing the DNA segments of interest can be transferredinto the host cell by well-known methods, depending on the type ofcellular host. For example, calcium chloride transfection is commonlyutilized for prokaryotic cells, whereas calcium phosphate treatment,electroporation, lipofection, biolistics or viral-based transfection canbe used for other cellular hosts. Other methods used to transformmammalian cells include the use of polybrene, protoplast fusion,liposomes, electroporation, and microinjection (see generally, Sambrooket al., supra). For production of transgenic animals, transgenes can bemicroinjected into fertilized oocytes, or can be incorporated into thegenome of embryonic stem cells, and the nuclei of such cells transferredinto enucleated oocytes.

Once expressed, antibodies can be purified according to standardprocedures of the art, including HPLC purification, columnchromatography, gel electrophoresis and the like (see generally, Scopes,Protein Purification (Springer-Verlag, NY, 1982)).

4. Other Therapeutic Agents

Therapeutic agents for use in the present methods also include T-cellsthat bind to Aβ peptide. For example, T-cells can be activated againstAβ peptide by expressing a human MHC class I gene and a humanβ-2-microglobulin gene from an insect cell line, whereby an emptycomplex is formed on the surface of the cells and can bind to Aβpeptide. T-cells contacted with the cell line become specificallyactivated against the peptide. See Peterson et al., U.S. Pat. No.5,314,813. Insect cell lines expressing an MHC class II antigen cansimilarly be used to activate CD4 T cells.

B. Other Diseases

The same or analogous principles determine production of therapeutic orpreventative agents for amyloidogenic diseases. In general, the agentsnoted above for use in treatment of Alzheimer's disease can also be usedfor treatment early onset Alzheimer's disease associated with Down'ssyndrome. In mad cow disease, prion peptide, active fragments, andanalogs, and antibodies to prion peptide are used in place of Aβpeptide, active fragments, analogs and antibodies to Aβ peptide intreatment of Alzheimer's disease. In treatment of multiple myeloma, IgGlight chain and analogs and antibodies thereto are used, and so forth inother diseases.

1. Carrier Proteins

Some agents for inducing an immune response contain the appropriateepitope for inducing an immune response against amyloid deposits but aretoo small to be immunogenic. In this situation, a peptide immunogen canbe linked to a suitable carrier to help elicit an immune response.Suitable carriers include serum albumins, keyhole limpet hemocyanin,immunoglobulin molecules, thyroglobulin, ovalbumin, tetanus toxoid, or atoxoid from other pathogenic bacteria, such as diphtheria, E. coli,cholera, or H. pylori, or an attenuated toxin derivative. Other carriersfor stimulating or enhancing an immune response include cytokines suchas IL-1, IL-1 α and β peptides, IL-2, ΓINF, IL-10, GM-CSF, andchemokines, such as MIP1α and β and RANTES. Immunogenic agents can alsobe linked to peptides that enhance transport across tissues, asdescribed in O'Mahony, WO 97/17613 and WO 97/17614.

Immunogenic agents can be linked to carriers by chemical crosslinking.Techniques for linking an immunogen to a carrier include the formationof disulfide linkages using N-succinimidyl-3-(2-pyridyl-thio) propionate(SPDP) and succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate(SMCC) (if the peptide lacks a sulfhydryl group, this can be provided byaddition of a cysteine residue). These reagents create a disulfidelinkage between themselves and peptide cysteine resides on one proteinand an amide linkage through the å-amino on a lysine, or other freeamino group in other amino acids. A variety of suchdisulfide/amide-forming agents are described by Immun. Rev. 62, 185(1982). Other bifunctional coupling agents form a thioether rather thana disulfide linkage. Many of these thio-ether-forming agents arecommercially available and include reactive esters of 6-maleimidocaproicacid, 2-bromoacetic acid, and 2-iodoacetic acid,4-(N-maleimido-methyl)cyclohexane-1-carboxylic acid. The carboxyl groupscan be activated by combining them with succinimide or1-hydroxyl-2-nitro-4-sulfonic acid, sodium salt.

Immunogenic peptides can also be expressed as fusion proteins withcarriers. The immunogenic peptide can be linked at the amino terminus,the carboxyl terminus, or internally to the carrier. Optionally,multiple repeats of the immunogenic peptide can be present in the fusionprotein.

The same or similar carrier proteins and methods of linkage can be usedfor generating immunogens to be used in generation of antibodies againstAβ for use in passive immunization. For example, Aβ or a fragment linkedto a carrier can be administered to a laboratory animal in theproduction of monoclonal antibodies to Aβ.

4. Nucleic Acid Encoding Therapeutic Agents

Immune responses against amyloid deposits can also be induced byadministration of nucleic acids encoding Aβ peptide, other peptideimmunogens, or antibodies and their component chains used for passiveimmunization. Such nucleic acids can be DNA or RNA. A nucleic acidsegment encoding an immunogen is typically linked to regulatoryelements, such as a promoter and enhancer, that allow expression of theDNA segment in the intended target cells of a patient. For expression inblood cells, as is desirable for induction of an immune response,promoter and enhancer elements from light or heavy chain immunoglobulingenes or the CMV major intermediate early promoter and enhancer aresuitable to direct expression. The linked regulatory elements and codingsequences are often cloned into a vector. For administration ofdouble-chain antibodies, the two chains can be cloned in the same orseparate vectors.

A number of viral vector systems are available including retroviralsystems (see, e.g., Lawrie and Tumin, Cur. Opin. Genet. Develop. 3,102-109 (1993)); adenoviral vectors (see, e.g., Bett et al., J. Virol.67, 5911 (1993)); adeno-associated virus vectors (see, e.g., Zhou etal., J. Exp. Med. 179, 1867 (1994)), viral vectors from the pox familyincluding vaccinia virus and the avian pox viruses, viral vectors fromthe alpha virus genus such as those derived from Sindbis and SemlikiForest Viruses (see, e.g., Dubensky et al., J. Virol. 70, 508-519(1996)), and papillomaviruses (Ohe et al., Human Gene Therapy 6, 325-333(1995); Woo et al., WO 94/12629 and Xiao & Brandsma, Nucleic Acids. Res.24, 2630-2622 (1996)).

DNA encoding an immunogen, or a vector containing the same, can bepackaged into liposomes. Suitable lipids and related analogs aredescribed by U.S. Pat. Nos. 5,208,036, 5,264,618, 5,279,833 and5,283,185. Vectors and DNA encoding an immunogen can also be adsorbed toor associated with particulate carriers, examples of which includepolymethyl methacrylate polymers and polylactides andpoly(lactide-co-glycolides), see, e.g., McGee et al., J. Micro Encap.(1996).

Gene therapy vectors or naked DNA can be delivered in vivo byadministration to an individual patient, typically by systemicadministration (e.g., intravenous, intraperitoneal, nasal, gastric,intradermal, intramuscular, subdermal, or intracranial infusion) ortopical application,(see e.g., U.S. Pat. No. 5,399,346). DNA can also beadministered using a gene gun. See Xiao & Brandsma, supra. The DNAencoding an immunogen is precipitated onto the surface of microscopicmetal beads. The microprojectiles are accelerated with a shock wave orexpanding helium gas, and penetrate tissues to a depth of several celllayers. For example, The Accel™ Gene Delivery Device manufactured byAgacetus, Inc. Middleton Wis. is suitable. Alternatively, naked DNA canpass through skin into the blood stream simply by spotting the DNA ontoskin with chemical or mechanical irritation (see WO 95/05853).

In a further variation, vectors encoding immunogens can be delivered tocells ex vivo, such as cells explanted from an individual patient (e.g.,lymphocytes, bone marrow aspirates, tissue biopsy) or universal donorhematopoietic stem cells, followed by reimplantation of the cells into apatient, usually after selection for cells which have incorporated thevector.

III. Patients Amenable to Treatment

Patients amenable to treatment include individuals at risk of diseasebut not showing symptoms, as well as patients presently showingsymptoms. In the case of Alzheimer's disease, virtually anyone is atrisk of suffering from Alzheimer's disease if be or she lives longenough. Therefore, the present methods can be administeredprophylactically to the general population without any assessment of therisk of the subject patient. The present methods are especially usefulfor individuals who do have a known genetic risk of Alzheimer's disease.Such individuals include those having relatives who have experiencedthis disease, and those whose risk is determined by analysis of geneticor biochemical markers. Genetic markers of risk toward Alzheimer'sdisease include mutations in the APP gene, particularly mutations atposition 717 and positions 670 and 671 referred to as the Hardy andSwedish mutations respectively (see Hardy, TINS, supra). Other markersof risk are mutations in the presenilin genes, PS1 and PS2, and ApoE4,family history of AD, hypercholesterolemia or atherosclerosis.Individuals presently suffering from Alzheimer's disease can berecognized from characteristic dementia, as well as the presence of riskfactors described above. In addition, a number of diagnostic tests areavailable for identifying individuals who have AD. These includemeasurement of CSF tau and Aβ42 levels. Elevated tau and decreased Aβ42levels signify the presence of AD. Individuals suffering fromAlzheimer's disease can also be diagnosed by ADRDA criteria as discussedin the Examples section.

In asymptomatic patients, treatment can begin at any age (e.g., 10, 20,30). Usually, however, it is not necessary to begin treatment until apatient reaches 40, 50, 60 or 70. Treatment typically entails multipledosages over a period of time. Treatment can be monitored by assayingantibody, or activated T-cell or B-cell responses to the therapeuticagent (e.g., Aβ peptide) over time. If the response falls, a boosterdosage is indicated. In the case of potential Down's syndrome patients,treatment can begin antenatally by administering therapeutic agent tothe mother or shortly after birth.

IV. Treatment Regimes

In prophylactic applications, pharmaceutical compositions or medicantsare administered to a patient susceptible to, or otherwise at risk of, aparticular disease in an amount sufficient to eliminate or reduce therisk or delay the outset of the disease. In therapeutic applications,compositions or medicants are administered to a patient suspected of, oralready suffering from such a disease in an amount sufficient to cure,or at least partially arrest, the symptoms of the disease and itscomplications. An amount adequate to accomplish this is defined as atherapeutically- or pharmaceutically-effective dose. In bothprophylactic and therapeutic regimes, agents are usually administered inseveral dosages until a sufficient immune response has been achieved.Typically, the immune response is monitored and repeated dosages aregiven if the immune response starts to fade.

Effective doses of the compositions of the present invention, for thetreatment of the above described conditions vary depending upon manydifferent factors, including means of administration, target site,physiological state of the patient, whether the patient is human or ananimal, other medications administered, and whether treatment isprophylactic or therapeutic. Usually, the patient is a human, but insome diseases, such as mad cow disease, the patient can be a nonhumanmammal, such as a bovine. Treatment dosages need to be titrated tooptimize safety and efficacy. The amount of immunogen depends on whetheradjuvant is also administered, with higher dosages being required in theabsence of adjuvant. The amount of an immunogen for administrationsometimes varies from 1-500 μg per patient and more usually from 5-500μg per injection for human administration. Occasionally, a higher doseof 1-2 mg per injection is used. Typically about 10, 20, 50 or 100 μg isused for each human injection. The timing of injections can varysignificantly from once a day, to once a year, to once a decade. On anygiven day that a dosage of immunogen is given, the dosage is greaterthan 1 μg/patient and usually greater than 10 μg/patient if adjuvant is,also administered, and greater than 10 μg/patient and usually greaterthan 100 μg/patient in the absence of adjuvant. A typical regimenconsists of an immunization followed by booster injections at 6 weekintervals. Another regimen consists of an immunization followed bybooster injections 1, 2 and 12 months later. Another regimen entails aninjection every two months for life. Alternatively, booster injectionscan be on an irregular basis as indicated by monitoring of immuneresponse.

For passive immunization with an antibody, the dosage ranges from about0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg, of the host bodyweight. For example dosages can be 1 mg/kg body weight or 10 mg/kg bodyweight. An exemplary treatment regime entails administration once perevery two weeks or once a month or once every 3 to 6 months. In somemethods, two or more monoclonal antibodies with different bindingspecificities are administered simultaneously, in which case the dosageof each antibody administered falls within the ranges indicated.Antibody is usually administered on multiple occasions. Intervalsbetween single dosages can be weekly, monthly or yearly. Intervals canalso be irregular as indicated by measuring blood levels of antibody toAβ in the patient. Alternatively, antibody can be administered as asustained release formulation, in which case less frequentadministration is required. Dosage and frequency vary depending on thehalf-life of the antibody in the patient. In general, human antibodiesshow the longest half life, followed by humanized antibodies, chimericantibodies, and nonhuman antibodies. The dosage and frequency ofadministration can vary depending on whether the treatment isprophylactic or therapeutic. In prophylactic applications, a relativelylow dosage is administered at relatively infrequent intervals over along period of time. Some patients continue to receive treatment for therest of their lives. In therapeutic applications, a relatively highdosage at relatively short intervals is sometimes required untilprogression of the disease is reduced or terminated, and preferablyuntil the patient shows partial or complete amelioration of symptoms ofdisease. Thereafter, the patent can be administered a prophylacticregime.

Doses for nucleic acids encoding immunogens range from about 10 ng to 1g, 100 ng to 100 mg, 1 μg to 10 mg, or 30-300 μg DNA per patient. Dosesfor infectious viral vectors vary from 10-109, or more, virions perdose.

Agents for inducing an immune response can be administered byparenteral, topical, intravenous, oral, subcutaneous, intraperitoneal,intranasal or intramuscular means for prophylactic and/or therapeutictreatment. The most typical route of administration of an immunogenicagent is subcutaneous although others can be equally effective. The nextmost common is intramuscular injection. This type of injection is mosttypically performed in the arm or leg muscles. Intravenous injections aswell as intraperitoneal injections, intraarterial, intracranial, orintradermal injections are also effective in generating an immuneresponse. In some methods, agents are injected directly into aparticular tissue where deposits have accumulated, for exampleintracranial injection. Intramuscular injection on intravenous infusionare preferred for administration of antibody. In some methods,particular therapeutic antibodies are injected directly into thecranium. In some methods, antibodies are administered as a sustainedrelease composition or device, such as a Medipad™ device.

Agents of the invention can optionally be administered in combinationwith other agents that are at least partly effective in treatment ofamyloidogenic disease. In the case of Alzheimer's and Down's syndrome,in which amyloid deposits occur in the brain, agents of the inventioncan also be administered in conjunction with other agents that increasepassage of the agents of the invention across the blood-brain barrier.

Immunogenic agents of the invention, such as peptides, are sometimesadministered in combination with an adjuvant. A variety of adjuvants canbe used in combination with a peptide, such as Aβ, to elicit an immuneresponse. Preferred adjuvants augment the intrinsic response to animmunogen without causing conformational changes in the immunogen thataffect the qualitative form of the response. Preferred adjuvants includealum, 3 De-O-acylated monophosphoryl lipid A (MPL) (see GB 2220211).QS-21 is a triterpene glycoside or saponin isolated from the bark of theQuillaja Saponaria Molina tree found in South America (see Kensil etal., in Vaccine Design: The Subunit and Ajuvant Approach (eds. Powell &Newman, Plenum Press, NY, 1995); U.S. Pat. No. 5,057,540). Otheradjuvants are oil in water emulsions (such as squalene or peanut oil),optionally in combination with immune stimulants, such as monophosphoryllipid A (see Stoute et al., N. Engl. J. Med. 336, 86-91 (1997)). Anotheradjuvant is CpG (Bioworld Today, Nov. 15, 1998). Alternatively, Aβ canbe coupled to an adjuvant. However, such coupling should notsubstantially change the conformation of Aβ so as to affect the natureof the immune response thereto. Adjuvants can be administered as acomponent of a therapeutic composition with an active agent or can beadministered separately, before, concurrently with, or afteradministration of the therapeutic agent.

A preferred class of adjuvants is aluminum salts (alum), such asaluminum hydroxide, aluminum phosphate, aluminum sulfate. Such adjuvantscan be used with or without other specific immunostimulating agents suchas MPL or 3-DMP, QS-21, polymeric or monomeric amino acids such aspolyglutamic acid or polylysine. Another class of adjuvants isoil-in-water emulsion formulations. Such adjuvants can be used with orwithout other specific immunostimulating agents such as muramyl peptides(e.g., N-acetylmuramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine(MTP-PE),N-acetylglucsaminyl-N-acetylmuramyl-L-Al-D-isoglu-L-Ala-dipalmitoxypropylamide (DTP-DPP) theramide™), or other bacterial cell wallcomponents. Oil-in-water emulsions include (a) MF59 (WO 90/14837),containing 5% Squalene, 0.5% Tween 80, and 0.5% Span 85 (optionallycontaining various amounts of MTP-PE) formulated into submicronparticles using a microfluidizer such as Model 110Y microfluidizer(Microfluidics, Newton Mass.), (b) SAF, containing 10% Squalane, 0.4%Tween 80, 5% pluronic-blocked polymer L121, and thr-MDP, eithermicrofluidized into a submicron emulsion or vortexed to generate alarger particle size emulsion, and (c) Ribi™ adjuvant system (RAS),(Ribi Immunochem, Hamilton, Mont.) containing 2% squalene, 0.2% Tween80, and one or more bacterial cell wall components from the groupconsisting of monophosphorylipid A (MPL), trehalose dimycolate (TDM),and cell wall skeleton (CWS), preferably MPL+CWS (Detox™). Another classof preferred adjuvants is saponin adjuvants, such as Stimulon™ (QS-21,Aquila, Worcester, Mass.) or particles generated therefrom such asISCOMs (immunostimulating complexes) and ISCOMATRIX. Other adjuvantsinclude Complete Freund's Adjuvant (CFA) and Incomplete Freund'sAdjuvant (IFA). Other adjuvants include cytokines, such as interleukins(IL-1,IL-2, and IL-12), macrophage colony stimulating factor (M-CSF),tumor necrosis factor (TNF).

An adjuvant can be administered with an immunogen as a singlecomposition, or can be administered before, concurrent with or afteradministration of the immunogen. Immunogen and adjuvant can be packagedand supplied in the same vial or can be packaged in separate vials andmixed before use. Immunogen and adjuvant are typically packaged with alabel indicating the intended therapeutic application. If immunogen andadjuvant are packaged separately, the packaging typically includesinstructions for mixing before use. The choice of an adjuvant and/orcarrier depends on the stability of the vaccine containing the adjuvant,the route of administration, the dosing schedule, the efficacy of theadjuvant for the species being vaccinated, and, in humans, apharmaceutically acceptable adjuvant is one that has been approved or isapprovable for human administration by pertinent regulatory bodies. Forexample, Complete Freund's adjuvant is not suitable for humanadministration. Alum, MPL and QS-21 are preferred. Optionally, two ormore different adjuvants can be used simultaneously. Preferredcombinations include alum with MPL, alum with QS-21, MPL with QS-21, andalum, QS-21 and MPL together. Also, Incomplete Freund's adjuvant can beused (Chang et al., Advanced Drug Delivery Reviews 32, 173-186 (1998)),optionally in combination with any of alum, QS-21, and MPL and allcombinations thereof.

Agents of the invention are often administered as pharmaceuticalcompositions comprising an active therapeutic agent, i.e., and a varietyof other pharmaceutically acceptable components. See Remington'sPharmaceutical Science (15th ed., Mack Publishing Company, Easton, Pa.,1980). The preferred form depends on the intended mode of administrationand therapeutic application. The compositions can also include,depending on the formulation desired, pharmaceutically-acceptable,non-toxic carriers or diluents, which are defined as vehicles commonlyused to formulate pharmaceutical compositions for animal or humanadministration. The diluent is selected so as not to affect thebiological activity of the combination. Examples of such diluents aredistilled water, physiological phosphate-buffered saline, Ringer'ssolutions, dextrose solution, and Hank's solution. In addition, thepharmaceutical composition or formulation may also include othercarriers, adjuvants, or nontoxic, nontherapeutic, nonimmunogenicstabilizers and the like. However, some reagents suitable foradministration to animals, such as Complete Freund's adjuvant are nottypically included in compositions for human use.

Pharmaceutical compositions can also include large, slowly metabolizedmacromolecules such as proteins, polysaccharides such as chitosan,polylactic acids, polyglycolic acids and copolymers (such as latexfunctionalized sepharose, agarose, cellulose, and the like), polymericamino acids, amino acid copolymers, and lipid aggregates (such as oildroplets or liposomes). Additionally, these carriers can function asimmunostimulating agents (i.e., adjuvants).

For parenteral administration, agents of the invention can beadministered as injectable dosages of a solution or suspension of thesubstance in a physiologically acceptable diluent with a pharmaceuticalcarrier that can be a sterile liquid such as water oils, saline,glycerol, or ethanol. Additionally, auxiliary substances, such aswetting or emulsifying agents, surfactants, pH buffering substances andthe like can be present in compositions. Other components ofpharmaceutical compositions are those of petroleum, animal, vegetable,or synthetic origin, for example, peanut oil, soybean oil, and mineraloil. In general, glycols such as propylene glycol or polyethylene glycolare preferred liquid carriers, particularly for injectable solutions.Antibodies can be administered in the form of a depot injection orimplant preparation which can be formulated in such a manner as topermit a sustained release of the active ingredient. An exemplarycomposition comprises monoclonal antibody at 5 mg/mL, formulated inaqueous buffer consisting of 50 mM L-histidine, 150 mM NaCl, adjusted topH 6.0 with HCl.

Typically, compositions are prepared as injectables, either as liquidsolutions or suspensions; solid forms suitable for solution in, orsuspension in, liquid vehicles prior to injection can also be prepared.The preparation also can be emulsified or encapsulated in liposomes ormicro particles such as polylactide, polyglycolide, or copolymer forenhanced adjuvant effect, as discussed above (see Langer, Science 249,1527 (1990) and Hanes, Advanced Drug Delivery Reviews 28, 97-119 (1997).The agents of this invention can be administered in the form of a depotinjection or implant preparation which can be formulated in such amanner as to permit a sustained or pulsatile release of the activeingredient.

Additional formulations suitable for other modes of administrationinclude oral, intranasal, and pulmonary formulations, suppositories, andtransdermal applications.

For suppositories, binders and carriers include, for example,polyalkylene glycols or triglycerides; such suppositories can be formedfrom mixtures containing the active ingredient in the range of 0.5% to10%, preferably 1%-2%. Oral formulations include excipients, such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, and magnesium carbonate. Thesecompositions take the form of solutions, suspensions, tablets, pills,capsules, sustained release formulations or powders and contain 10%-95%of active ingredient, preferably 25%-70%.

Topical application can result in transdermal or intradermal delivery.Topical administration can be facilitated by co-administration of theagent with cholera toxin or detoxified derivatives or subunits thereofor other similar bacterial toxins (See Glenn et al., Nature 391, 851(1998)). Co-administration can be achieved by using the components as amixture or as linked molecules obtained by chemical crosslinking orexpression as a fusion protein.

Alternatively, transdermal delivery can be achieved using a skin path orusing transferosomes (Paul et al., Eur. J. Immunol. 25, 3521-24 (1995);Cevc et al., Biochem. Biophys. Acta 1368, 201-15 (1998)).

V. Methods of Diagnosis

The invention provides methods of detecting an immune response againstAβ peptide in a patient suffering from or susceptible to Alzheimer'sdisease. The methods are particularly useful for monitoring a course oftreatment being administered to a patient. The methods can be used tomonitor both therapeutic treatment on symptomatic patients andprophylactic treatment on asymptomatic patients. The methods are usefulfor monitoring both active immunization (e.g., antibody produced inresponse to administration of immunogen) and passive immunization (e.g.,measuring level of administered antibody).

1. Active Immunization

Some methods entail determining a baseline value of an immune responsein a patient before administering a dosage of agent, and comparing thiswith a value for the immune response after treatment. A significantincrease (i.e., greater than the typical margin of experimental error inrepeat measurements of the same sample, expressed as one standarddeviation from the mean of such measurements) in value of the immuneresponse signals a positive treatment outcome (i.e., that administrationof the agent has achieved or augmented an immune response). If the valuefor immune response does not change significantly, or decreases, anegative treatment outcome is indicated. In general, patients undergoingan initial course of treatment with an immunogenic agent are expected toshow an increase in immune response with successive dosages, whicheventually reaches a plateau. Administration of agent is generallycontinued while the immune response is increasing. Attainment of theplateau is an indicator that the administered of treatment can bediscontinued or reduced in dosage or frequency.

In other methods, a control value (i.e., a mean and standard deviation)of immune response is determined for a control population. Typically theindividuals in the control population have not received prior treatment.Measured values of immune response in a patient after administering atherapeutic agent are then compared with the control value. Asignificant increase relative to the control value (e.g., greater thanone standard deviation from the mean) signals a positive treatmentoutcome. A lack of significant increase or a decrease signals a negativetreatment outcome. Administration of agent is generally continued whilethe immune response is increasing relative to the control value. Asbefore, attainment of a plateau relative to control values in anindicator that the administration of treatment can be discontinued orreduced in dosage or frequency.

In other methods, a control value of immune response (e.g., a mean andstandard deviation) is determined from a control population ofindividuals who have undergone treatment with a therapeutic agent andwhose immune responses have plateaued in response to treatment. Measuredvalues of immune response in a patient are compared with the controlvalue. If the measured level in a patient is not significantly different(e.g., more than one standard deviation) from the control value,treatment can be discontinued. If the level in a patient issignificantly below the control value, continued administration of agentis warranted. If the level in the patient persists below the controlvalue, then a change in treatment regime, for example, use of adifferent adjuvant may be indicated.

In other methods, a patient who is not presently receiving treatment buthas undergone a previous course of treatment is monitored for immuneresponse to determine whether a resumption of treatment is required. Themeasured value of immune response in the patient can be compared with avalue of immune response previously achieved in the patient after aprevious course of treatment. A significant decrease relative to theprevious measurement (i.e., greater than a typical margin of error inrepeat measurements of the same sample) is an indication that treatmentcan be resumed. Alternatively, the value measured in patient can becompared with a control value (mean plus standard deviation) determinedin population of patients after undergoing a course of treatment.Alternatively, the measured value in a patient can be compared with acontrol value in populations of prophylactically treated patients whoremain free of symptoms of disease, or populations of therapeuticallytreated patients who show amelioration of disease characteristics. Inall of these cases, a significant decrease relative to the control level(i.e., more than a standard deviation) is an indicator that treatmentshould be resumed in a patient.

The tissue sample for analysis is typically blood, plasma, serum, mucousor cerebrospinal fluid from the patient. The sample is analyzed forindication of an immune response to any form of Aβ peptide, typicallyAβ42. The immune response can be determined from the presence of, e.g.,antibodies or T-cells that specifically bind to Aβ peptide. ELISAmethods of detecting antibodies specific to Aβ are described in theExamples section. Methods of detecting reactive T-cells have beendescribed above (see Definitions).

2. Passive Immunization

In general, the procedures for monitoring passive immunization aresimilar to those for monitoring active immunization described above.However, the antibody profile following passive immunization typicallyshows an immediate peak in antibody concentration followed by anexponential decay. Without a further dosage, the decay approachespretreatment levels within a period of days to months depending on thehalf-life of the antibody administered. For example the half-life ofsome human antibodies is of the order of 20 days.

In some methods, a baseline measurement of antibody to Aβ in the patientis made before administration, a second measurement is made soonthereafter to determine the peak antibody level, and one or more furthermeasurements are made at intervals to monitor decay of antibody levels.When the level of antibody has declined to baseline or a predeterminedpercentage of the peak less baseline (e.g., 50%, 25% or 10%),administration of a further dosage of antibody is administered. In somemethods, peak or subsequent measured levels less background are comparedwith reference levels previously determined to constitute a beneficialprophylactic or therapeutic treatment regime in other patients. If themeasured antibody level is significantly less than a reference level(e.g., less than the mean minus one standard deviation of the referencevalue in population of patients benefiting from treatment)administration of an additional dosage of antibody is indicated.

3. Diagnostic Kits

The invention further provides diagnostic kits for performing thediagnostic methods described above. Typically, such kits contain anagent that specifically binds to antibodies to Aβ or reacts withT-cells-specific for Aβ. The kit can also include a label. For detectionof antibodies to Aβ, the label is typically in the form of labelledanti-idiotypic antibodies. For detection of antibodies, the agent can besupplied prebound to a solid phase, such as to the wells of a microtiterdish. For detection of reactive T-cells, the label can be supplied as3H-thymidine to measure a proliferative response. Kits also typicallycontain labelling providing directions for use of the kit. The labellingmay also include a chart or other correspondence regime correlatinglevels of measured label with levels of antibodies to Aβ or T-cellsreactive with Aβ. The term labelling refers to any written or recordedmaterial that is attached to, or otherwise accompanies a kit at any timeduring its manufacture, transport, sale or use. For example, the termlabelling encompasses advertising leaflets and brochures, packagingmaterials, instructions, audio or video cassettes, computer discs, aswell as writing imprinted directly on kits.

EXAMPLES

I. Prophylactic Efficacy of Aβ Against AD

These examples describe administration of Aβ42 peptide to transgenicmice overexpressing APP with a mutation at position 717 (APP_(717V→F))that predisposes them to develop Alzheimer's-like neuropathology.Production and characteristics of these mice (PDAPP mice) is describedin Games et al., Nature, supra. These animals, in their heterozygoteform, begin to deposit Aβ at six months of age forward. By fifteenmonths of age they exhibit levels of Aβ deposition equivalent to thatseen in Alzheimer's disease. PDAPP mice were injected with aggregatedAβ₄₂ (aggregated Aβ₄₂) or phosphate buffered saline. Aggregated Aβ₄₂ waschosen because of its ability to induce antibodies to multiple epitopesof Aβ.

A. Methods

1. Source of Mice

Thirty PDAPP heterogenic female mice were randomly divided into thefollowing groups: 10 mice to be injected with aggregated Aβ42 (one diedin transit), 5 mice to be injected with PBS/adjuvant or PBS, and 10uninjected controls. Five mice were injected with peptides dervied fromthe sequence of serum amyloid protein (SAP).

2. Preparation of Immunogens

Preparation of aggregated Aβ42: two milligrams of Aβ42 (US Peptides Inc,lot K-42-12) was dissolved in 0.9 ml water and made up to 1 ml by adding0.1 ml 10×PBS. This was vortexed and allowed to incubate overnight 37°C., under which conditions the peptide aggregated. Any unused Aβ wasstored as a dry lyophilized powder at −20° C. until the next injection.

3. Preparation of Injections

For each injection, 100 μg of aggregated Aβ42 in PBS per mouse wasemulsified 1:1 with Complete Freund's adjuvant (CFA) in a final volumeof 400 μl emulsion for the first immunization, followed by a boost ofthe same amount of immunogen in Incomplete Freund's adjuvant (IFA) at 2weeks. Two additional doses in IFA were given at monthly intervals. Thesubsequent immunizations were done at monthly intervals in 500 μl ofPBS. Injections were delivered intraperitoneally (i.p.).

PBS injections followed the same schedule and mice were injected with a1:1 mix of PBS/Adjuvant at 400 μl per mouse, or 500 μl of PBS per mouse.SAP injections likewise followed the same schedule using a dose of 100μg per injection.

4. Titration of Mouse Bleeds, Tissue Preparation andImmunohistochemistry

The above methods are described infra in General Materials and Methods.

B. Results

PDAPP mice were injected with either aggregated Aβ42 (aggregated Aβ42),SAP peptides, or phosphate buffered saline. A group of PDAPP mice werealso left as uninjected, positive controls. The titers of the mice toaggregated Aβ42 were monitored every other month from the fourth boostuntil the mice were one year of age. Mice were sacrificed at 13 months.At all time points examined, eight of the nine aggregated Aβ42 micedeveloped a high antibody titer, which remained high throughout theseries of injections (titers greater than 1/10000). The ninth mouse hada low, but measurable titer of approximately 1/1000 (FIG. 1, Table 1).SAPP-injected mice had titers of 1:1,000 to 1:30,000 for this immunogenwith only a single mouse exceeding 1:10,0000.

The PBS-treated mice were titered against aggregated Aβ42 at six, tenand twelve months. At a 1/100 dilution the PBS mice when titered againstaggregated Aβ42 only exceeded 4 times background at one data point,otherwise, they were less than 4 times background at all time points(Table 1). The SAP-specific response was negligible at these time pointswith all titers less than 300.

Seven out of the nine mice in the aggregated Aβ1-42 treated group had nodetectable amyloid in their brains. In contrast, brain tissue from micein the SAP and PBS groups contained numerous amyloid deposits in thehippocampus, as well as in the frontal and cingulate cortices. Thepattern of deposition was similar to that of untreated controls, withcharacteristic involvement of vulnerable subregions, such as the outermolecular layer of the hippocampal dentate gyrus. One mouse from the Aβ1-42-injected group had a greatly reduced amyloid burden, confined tothe hippocampus. An isolated plaque was identified in another Aβ1-42-treated mouse.

Quantitative image analyses of the amyloid burden in the hippocampusverified the dramatic reduction achieved in the Aβ42(AN1792)-treatedanimals (FIG. 2). The median values of the amyloid burden for the PBSgroup (2.22%), and for the untreated control group (2.65%) weresignificantly greater than for those immunized with AN1792 (0.00%,p=0.0005). In contrast, the median value for the group immunized withSAP peptides (SAPP) was 5.74%. Brain tissue from the untreated, controlmice contained numerous Aβ amyloid deposits visualized with theAβ-specific monoclonal antibody (mAb) 3D6 in the hippocampus, as well asin the retrosplenial cortex. A similar pattern of amyloid deposition wasalso seen in mice immunized with SAPP or PBS (FIG. 2). In addition, inthese latter three groups there was a characteristic involvement ofvulnerable subregions of the brain classically seen in AD, such as theouter molecular layer of the hippocampal dentate gyrus, in all three ofthese groups.

The brains that contained no Aβ deposits were also devoid of neuriticplaques that are typically visualized in PDAPP mice with the human APPantibody 8E5. All of brains from the remaining groups (SAP-injected, PBSand uninjected mice) had numerous neuritic plaques typical of untreatedPDAPP mice. A small number of neuritic plaques were present in one mousetreated with AN1792, and a single cluster of dystrophic neurites wasfound in a second mouse treated with AN1792. Image analyses of thehippocampus, and shown in FIG. 3, demonstrated the virtual eliminationof dystrophic neurites in AN1792-treated mice (median 0.00%) compared tothe PBS recipients (median 0.28%, p=0.0005).

Astrocytosis characteristic of plaque-associated inflammation was alsoabsent in the brains of the Aβ1-42 injected group. The brains from themice in the other groups contained abundant and clustered GFAP-positiveastrocytes typical of Aβ plaque-associated gliosis. A subset of theGFAP-reacted slides were counter-stained with Thioflavin S to localizethe Aβ deposits. The GFAP-positive astrocytes were associated with Aβplaques in the SAP, PBS and untreated controls. No such association wasfound in the plaque-negative Aβ1-42 treated mice, while minimalplaque-associated gliosis was identified in one mouse treated withAN1792.

Image analyses, shown in FIG. 4 for the retrosplenial cortex, verifiedthat the reduction in astrocytosis was significant with a median valueof 1.56% for those treated with AN1792 versus median values greater than6% for groups immunized with SAP peptides, PBS or untreated (p=0.0017)

Evidence from a subset of the Aβ1-42- and PBS-injected mice indicatedplaque-associated MHC II immunoreactivity was absent in the AβI-42injected mice, consistent with lack of an Aβ-related inflammatoryresponse.

Sections of the mouse brains were also reacted with a mAb specific witha monoclonal antibody specific for MAC-1, a cell surface protein. MAC-1(CD11b) is an integrin family member and exists as a heterodimer withCD18. The CD11b/CD18 complex is present on monocytes, macrophages,neutrophils and natural killer cells (Mak and Simard). The residentMAC-1-reactive cell type in the brain is likely to be microglia based onsimilar phenotypic morphology in MAC-1 immunoreacted sections.Plaque-associated MAC-1 labeling was lower in the brains of mice treatedwith AN1792 compared to the PBS control group, a finding consistent withthe lack of an Aβ-induced inflammatory response.

C. Conclusion

The lack of Aβ plaques and reactive neuronal and gliotic changes in thebrains of the Aβ1-42-injected mice indicate that no or extremely littleamyloid was deposited in their brains, and pathological consequences,such as gliosis and neuritic pathology, were absent. PDAPP mice treatedwith Aβ1-42 show essentially the same lack of pathology as controlnontransgenic mice. Therefore, Aβ1-42 injections are highly effective inthe prevention of deposition or clearance of human Aβ from brain tissue,and elimination of subsequent neuronal and inflammatory degenerativechanges. Thus, administration of Aβ peptide can have both preventativeand therapeutic benefit in prevention of AD.

II. Dose Response Study

Groups of five-week old, female Swiss Webster mice (N=6 per group) wereimmunized with 300, 100, 33, 11, 3.7, 1.2, 0.4, or 0.13 ug of Aβformulated in CFA/IFA administered intraperitoneally. Three doses weregiven at biweekly intervals followed by a fourth dose one month later.The first dose was emulsified with CFA and the remaining doses wereemulsified with IFA. Animals were bled 4-7 days following eachimmunization starting after the second dose for measurement of antibodytiters. Animals in a subset of three groups, those immunized with 11,33, or 300 μg of antigen, were additionally bled at approximatelymonthly intervals for four months following the fourth immunization tomonitor the decay of the antibody response across a range of vaccinedoses. These animals received a final fifth immunization at seven monthsafter study initiation. They were sacrificed one week later to measureantibody responses to AN1792 and to perform toxicological analyses.

A declining dose response was observed from 300 to 3.7 μg with noresponse at the two lowest doses. Mean antibody titers are about 1:1000after 3 doses and about 1:10,000 after 4 doses of 11-300 μg of antigen(see FIG. 5).

Antibody titers rose dramatically for all but the lowest dose groupfollowing the third immunization with increases in GMTs ranging from 5-to 25-fold. Low antibody responses were then detectable for even the 0.4μg recipients. The 1.2 and 3.7 μg groups had comparable titers with GMTsof about 1000 and the highest four doses clustered together with GMTs ofabout 25,000, with the exception of the 33 μg dose group with a lowerGMT of 3000. Following the fourth immunization, the titer increase wasmore modest for most groups. There was a clear dose response across thelower antigen dose groups from 0.14 μg to 11 μg ranging from nodetectable antibody for recipients of 0.14 μg to a GMT of 36,000 forrecipients of 11 μg. Again, titers for the four highest dose groups of11 to 300 μg clustered together. Thus following two immunizations, theantibody titer was dependent on the antigen dose across the broad rangefrom 0.4 to 300 μg. By the third immunization, titers of the highestfour doses were all comparable and they remained at a plateau after anadditional immunization.

One month following the fourth immunization, titers were 2- to 3-foldhigher in the 300 μg group than those measured from blood drawn fivedays following the immunization (FIG. 6). This observation suggests thatthe peak anamnestic antibody response occurred later than 5 dayspost-immunization. A more modest (50%) increase was seen at this time inthe 33 μg group. In the 300 μg dose group at two months following thelast dose, GMTs declined steeply by about 70%. After another month, thedecline was less steep at 45% (100 μg) and about 14% for the 33 and 11μg doses. Thus, the rate of decline in circulating antibody titersfollowing cessation of immunization appears to be biphasic with a steepdecline the first month following peak response followed by a moremodest rate of decrease thereafter.

The antibody titers and the kinetics of the response of these SwissWebster mice are similar to those of young heterozygous PDAPP transgenicmice immunized in a parallel manner. Dosages effective to induce animmune response in humans are typically similar to dosages effective inmice.

III. Screen for Therapeutic Efficacy Against Established AD

This assay is designed to test immunogenic agents for activity inarresting or reversing neuropathologic characteristics of AD in agedanimals. Immunizations with 42 amino acid long Aβ (AN1792) were begun ata time point when amyloid plaques are already present in the brains ofthe PDAPP mice.

Over the time course used in this study, untreated PDAPP mice develop anumber of neurodegenerative changes that resemble those found in AD(Games et al, supra and Johnson-Wood et al., Proc. Natl. Acad. Sci. USA94, 1550-1555 (1997)). The deposition of Aβ into amyloid plaques isassociated with a degenerative neuronal response consisting of aberrantaxonal and dendritic elements, called dystrophic neurites. Amyloiddeposits that are surrounded by and contain dystrophic neurites calledneuritic plaques. In both AD and the PDAPP mouse, dystrophic neuriteshave a distinctive globular structure, are immunoreactive with a panelof antibodies recognizing APP and cytoskeletal components, and displaycomplex subcellular degenerative changes at the ultrastructural level.These characteristics allow for disease-relevant, selective andreproducible measurements of neuritic plaque formation in the PDAPPbrains. The dystrophic neuronal component of PDAPP neuritic plaques iseasily visualized with an antibody specific for human APP (monoclonalantibody 8E5), and is readily measurable by computer-assisted imageanalysis. Therefore, in addition to measuring the effects of AN1792 onamyloid plaque formation, we monitored the effects of this treatment onthe development of neuritic dystrophy.

Astrocytes and microglia are non-neuronal cells that respond to andreflect the degree of neuronal injury. GFAP-positive astrocytes and MHCII-positive microglia are commonly observed in AD, and their activationincreases with the severity of the disease. Therefore, we also monitoredthe development of reactive astrocytosis and microgliosis in theAN1792-treated mice.

A. Materials and Methods

Forty-eight, heterozygous female PDAPP mice, 11 to 11.5 months of age,obtained from Charles River, were randomly divided into two groups: 24mice to be immunized with 100 μg of AN1792 and 24 mice to be immunizedwith PBS, each combined with Freund's adjuvant. The AN1792 and PBSgroups were again divided when they reached ˜15 months of age. At 15months of age approximately half of each group of the AN1792- andPBS-treated animals were euthanized (n=10 and 9, respectively), theremainder continued to receive immunizations until termination at ˜18months (n=9 and 12, respectively). A total of 8 animals (5 AN1792, 3PBS) died during the study. In addition to the immunized animals,one-year old (n=10), 15-month old (n=10) and 18-month old (n=10)untreated PDAPP mice were included for comparison in the ELISAs tomeasure aβ and APP levels in the brain; the one-year old animals werealso included in the immunohistochemical analyses.

Methodology was as in Example 1 unless otherwise indicated. US Peptideslot 12 and California Peptides lot ME0339 of AN1792 were used to preparethe antigen for the six immunizations administered prior to the 15-monthtime point. California Peptides lots ME0339 and ME0439 were used for thethree additional immunizations administered between 15 and 18 months.

For immunizations, 100μg of AN1792 in 200 μl PBS or PBS alone wasemulsified 1:1 (vol:vol) with Complete Freund's adjuvant (CFA) orIncomplete Freund's adjuvant (IFA) or PBS in a final volume of 400 μl.The first immunization was delivered with CFA as adjuvant, the next fourdoses were given with IFA and the final four doses with PBS alonewithout added adjuvant. A total of nine immunizations were given overthe seven-month period on a two-week schedule for the first three dosesfollowed by a four-week interval for the remaining injections. Thefour-month treatment group, euthanized at 15 months of age, receivedonly the first 6 immunizations.

B. Results

1. Effects of AN1792 Treatment on Amyloid Burden

The results of AN1792 treatment on cortical amyloid burden determined byquantitative image analysis are shown in FIG. 7. The median value ofcortical amyloid burden was 0.28% in a group of untreated 12-month oldPDAPP mice, a value representative of the plaque load in mice at thestudy's initiation. At 18 months, the amyloid burden increased over17-fold to 4.87% in PBS-treated mice, while AN1792-treated mice had agreatly reduced amyloid burden of only 0.01%, notably less than the12-month untreated and both the 15- and 18-month PBS-treated groups. Theamyloid burden was significantly reduced in the AN1792 recipients atboth 15 (96% reduction; p=0.003) and 18 (>99% reduction; p=0.0002)months.

Typically, cortical amyloid deposition in PDAPP mice initiates in thefrontal and retrosplenial cortices (RSC) and progresses in aventral-lateral direction to involve the temporal and entorhinalcortices (EC). Little or no amyloid was found in the EC of 12 month-oldmice, the approximate age at which AN1792 was first administered. After4 months of AN1792 treatment, amyloid deposition was greatly diminishedin the RSC, and the progressive involvement of the EC was entirelyeliminated by AN1792 treatment. The latter observation showed thatAN1792 completely halted the progression of amyloid that would normallyinvade the temporal and ventral cortices, as well as arrested orpossibly reversed deposition in the RSC.

The profound effects of AN1792 treatment on developing cortical amyloidburden in the PDAPP mice are further demonstrated by the 18-month group,which had been treated for seven months. A near complete absence ofcortical amyloid was found in the AN1792-treated mouse, with a totallack of diffuse plaques, as well as a reduction in compacted deposits.

2. AN1792 Treatment-Associated Cellular and Morphological

Changes

A population of Aβ-positive cells was found in brain regions thattypically contain amyloid deposits. Remarkably, in several brains fromAN1792 recipients, very few or no extracellular cortical amyloid plaqueswere found. Most of the Aβ immunoreactivity appeared to be containedwithin cells with large lobular or clumped soma. Phenotypically, thesecells resembled activated microglia or monocytes. They wereimmunoreactive with antibodies recognizing ligands expressed byactivated monocytes and microglia (MHC II and CD11b) and wereoccasionally associated with the wall or lumen of blood vessels.Comparison of near-adjacent sections labeled with Aβ and MHC II-specificantibodies revealed that similar patterns of these cells were recognizedby both classes of antibodies. Detailed examination of theAN1792-treated brains revealed that the MHC II-positive cells wererestricted to the vicinity of the limited amyloid remaining in theseanimals. Under the fixation conditions employed, the cells were notimmunoreactive with antibodies that recognize T cell (CD3, CD3e) or Bcell (CD45RA, CD45RB) ligands or leukocyte common antigen (CD45), butwere reactive with an antibody recognizing leukosialin (CD43) whichcross-reacts with monocytes. No such cells were found in any of thePBS-treated mice.

PDAPP mice invariably develop heavy amyloid deposition in the outermolecular layer of the hippocampal dentate gyrus. The deposition forms adistinct streak within the perforant pathway, a subregion thatclassically contains amyloid plaques in AD. The characteristicappearance of these deposits in PBS-treated mice resembled thatpreviously characterized in untreated PDAPP mice. The amyloid depositionconsisted of both diffuse and compacted plaques in a continuous band. Incontrast, in a number of brains from AN1792-treated mice this patternwas drastically altered. The hippocampal amyloid deposition no longercontained diffuse amyloid, and the banded pattern was completelydisrupted. Instead, a number of unusual punctate structures were presentthat are reactive with anti-Aβ antibodies, several of which appeared tobe amyloid-containing cells.

MHC II-positive cells were frequently observed in the vicinity ofextracellular amyloid in AN1792-treated animals. The pattern ofassociation of Aβ-positive cells with amyloid was very similar inseveral brains from AN1792-treated mice. The distribution of thesemonocytic cells was restricted to the proximity of the deposited amyloidand was entirely absent from other brain regions devoid of Aβ plaques.

Quantitative image analysis of MHC II and MAC I-labeled sectionsrevealed a trend towards increased immunoreactivity in the RSC andhippocampus of AN1792-treated mice compared to the PBS group whichreached significance with the measure of MAC 1 reactivity inhippocampus.

These results are indicative of active, cell-mediated removal of amyloidin plaque-bearing brain regions.

3. AN1792 Effects on Aβ Levels: ELISA Determinations

(a) Cortical Levels

In untreated PDAPP mice, the median level of total Aβ in the cortex at12 months was 1,600 ng/g, which increased to 8,700 ng/g by 15 months(Table 2). At 18 months the value was 22,000 ng/g, an increase of over10-fold during the time course of the experiment. PBS-treated animalshad 8,600 ng/g total Aβ at 15 months which increased to 19,000 ng/g at18 months. In contrast, AN1792-treated animals had 81% less total Aβ at15 months (1,600 ng/g) than the PBS-immunized group. Significantly less(p=0.0001) total Aβ (5,200 ng/g) was found at 18 months when the AN1792and PBS groups were compared (Table 2), representing a 72% reduction inthe Aβ that would otherwise be present. Similar results were obtainedwhen cortical levels of Aβ42 were compared, namely that the AN1792-treated group contained much less Aβ42, but in this case thedifferences between the AN1792 and PBS groups were significant at both15 months (p=0.04) and 18 months (p=0.0001, Table 2). TABLE 2 Median AβLevels (ng/g) in Cortex UNTREATED PBS AN1792 Age Total Aβ42 (n) TotalAβ42 (n) Total Aβ42 (n) 12 1,600 1,300 (10) 15 8,700 8,300 (10) 8,6007,200  (9) 1,600 1,300*  (10) 18 22,200 18,500 (10) 19,000 15,900 (12) 5,200** 4,000**  (9)*p = 0.0412**p = 0.0001

(b) Hippocampal Levels

In untreated PDAPP mice, median hippocampal levels of total Aβ at twelvemonths of age were 15,000 ng/g which increased to 51,000 ng/g at 15months and further to 81,000 ng/g at 18 months (Table 3). Similarly, PBSimmunized mice showed values of 40,000 ng/g and 65,000 ng/g at 15 monthsand 18 months, respectively. AN1792 immunized animals exhibited lesstotal Aβ, specifically 25,000 ng/g and 51,000 ng/g at the respective15-month and 18-month timepoints. The 18-month AN1792-treated groupvalue was significantly lower than that of the PBS treated group(p=0.0105; Table 3). Measurement of Aβ42 gave the same pattern ofresults, namely that levels in the AN1792-treated group weresignificantly lower than in the PBS group (39,000 ng/g vs. 57,000 ng/g,respectively; p=0.002) at the 18-month evaluation (Table 3). TABLE 3Median Aβ Levels (ng/g) in Hippocampus UNTREATED PBS AN1792 Age TotalAβ42 (n) Total Aβ42 (n) Total Aβ42 (n) 12 15,500 11,100 (10) 15 51,50044,400 (10) 40,100 35.70  (9) 24.50 22,100  (10) 18 80,800 64,200 (10)65,400 57.10 (12) 50.90 38,900**  (9)*p = 0.0105**p = 0.0022

(c) Cerebellar Levels

In 12-month untreated PDAPP mice, the median cerebellar level of totalAβ was 15 ng/g (Table 4). At 15 months, this median increased to 28 ng/gand by 18 months had risen to 35 ng/g. PBS-treated animals displayedmedian total Aβ values of 21 ng/g at 15 months and 43 ng/g at 18 months.AN1792-treated animals were found to have 22 ng/g total Aβ at 15 monthsand significantly less (p=0.002) total Aβ at 18 months (25 ng/g) thanthe corresponding PBS group (Table 4). TABLE 4 Median Aβ Levels (ng/g)in Cerebellum UNTREATED PBS AN1792 Age Total Aβ (n) Total Aβ (n) TotalAβ (n) 12 15.6 (10) 15 27.7 (10) 20.8  (9) 21.7  (10) 18 35.0 (10) 43.1(12) 24.8*  (9)*p = 0.0018

4. Effects of AN1792 Treatment on APP Levels

APP-α and the full-length APP molecule both contain all or part of theAβ sequence and thus could be potentially impacted by the generation ofan AN1792-directed immune response. In studies to date, a slightincrease in APP levels has been noted as neuropathology increases in thePDAPP mouse. In the cortex, levels of either APP-á/FL (full length) orAPP-α were essentially unchanged by treatment with the exception thatAPP-á was reduced by 19% at the 18-month timepoint in the AN1792-treatedvs. the PBS-treated group. The 18-month AN1792-treated APP values werenot significantly different from values of the 12-month and 15-monthuntreated and 15-month PBS groups. In all cases the APP values remainedwithin the ranges that are normally found in PDAPP mice.

5. Effects of AN1792 Treatment on Neurodegenerative and GlioticPathology

Neuritic plaque burden was significantly reduced in the frontal cortexof AN1792-treated mice compared to the PBS group at both 15 (84%;p=0.03) and 18 (55%; p=0.01) months of age (FIG. 8). The median value ofthe neuritic plaque burden increased from 0.32% to 0.49% in the PBSgroup between 15 and 18 months of age. This contrasted with the greatlyreduced development of neuritic plaques in the AN1792 group, with medianneuritic plaque burden values of 0.05% and 0.22%, in the 15 and 18 monthgroups, respectively.

Immunizations with AN1792 seemed well tolerated and reactiveastrocytosis was also significantly reduced in the RSC of AN1792-treatedmice when compared to the PBS group at both 15 (56%; p=0.011) and 18(39%; p=0.028) months of age (FIG. 9). Median values of the percent ofastrocytosis in the PBS group increased between 15 and 18 months from4.26% to 5.21%. AN1792-treatment suppressed the development ofastrocytosis at both time points to 1.89% and 3.2%, respectively. Thissuggests the neuropil was not being damaged by the clearance process.

6. Antibody Responses

As described above, eleven-month old, heterozygous PDAPP mice (N=24)received a series of 5 immunizations of 100 μg of AN1792 emulsified withFreund's adjuvant and administered intraperitoneally at weeks 0, 2, 4,8, and 12, and a sixth immunization with PBS alone (no Freund'sadjuvant) at week 16. As a negative control, a parallel set of 24age-matched transgenic mice received immunizations of PBS emulsifiedwith the same adjuvants and delivered on the same schedule. Animals werebled within three to seven days following each immunization startingafter the second dose. Antibody responses to AN1792 were measured byELISA. Geometric mean titers (GMT) for the animals that were immunizedwith AN1792 were approximately 1,900, 7,600, and 45,000 following thesecond, third and last (sixth) doses respectively. No Aβ-specificantibody was measured in control animals following the sixthimmunization.

Approximately one-half of the animals were treated for an additionalthree months, receiving immunizations at about 20, 24 and 27 weeks. Eachof these doses was delivered in PBS vehicle alone without Freund'sadjuvant. Mean antibody titers remained unchanged over this time period.In fact, antibody titers appeared to remain stable from the fourth tothe eighth bleed corresponding to a period covering the fifth to theninth injections.

To determine if the Aβ-specific antibodies elicited by immunization thatwere detected in the sera of AN1792-treated mice were also associatedwith deposited brain amyloid, a subset of sections from the AN1792- andPBS-treated mice were reacted with an antibody specific for mouse IgG.In contrast to the PBS group, Aβ plaques in AN1792-treated brains werecoated with endogenous IgG. This difference between the two groups wasseen in both 15-and 18-month groups. Particularly striking was the lackof labeling in the PBS group, despite the presence of a heavy amyloidburden in these mice. These results show that immunization with asynthetic Aβ protein generates antibodies that recognize and bind invivo to the Aβ in amyloid plaques.

7. Cellular-Mediated Immune Responses

Spleens were removed from nine AN1792-immunized and 12 PBS-immunized18-month old PDAPP mice 7 days after the ninth immunization. Splenocyteswere isolated and cultured for 72 h in the presence of Aβ40, Aβ42, orAβ40-1 (reverse order protein). The mitogen Con A served as a positivecontrol. Optimum responses were obtained with >1.7 μM protein. Cellsfrom all nine AN1792-treated animals proliferated in response to eitherAβ1-40 or Aβ1-42 protein, with equal levels of incorporation for bothproteins (FIG. 10A). There was no response to the Aβ40-1 reverseprotein. Cells from control animals did not respond to any of the Aβproteins (FIG. 10B).

C. Conclusion

The results of this study show that AN1792 immunization of PDAPP micepossessing existing amyloid deposits slows and prevents progressiveamyloid deposition and retard consequential neuropathologic changes inthe aged PDAPP mouse brain. Immunizations with AN1792 essentially haltedamyloid developing in structures that would normally succumb toamyloidosis. Thus, administration of Aβ peptide has therapeutic benefitin the treatment of AD.

IV. Screen of Aβ Fragments

100 PDAPP mice age 9-11 months were immunized with 9 different regionsof APP and Aβ to determine which epitopes convey the efficaciousresponse. The 9 different immunogens and one control are injected i.p.as described above. The immunogens include four human Aβ peptideconjugates 1-12, 13-28, 32-42, 1-5, all coupled to sheep anti-mouse IgGvia a cystine link; an APP polypeptide amino acids 592-695, aggregatedhuman Aβ 1-40, and aggregated human Aβ 25-35, and aggregated rodentAβ42. Aggregated Aβ42 and PBS were used as positive and negativecontrols, respectively. Ten mice were used per treatment group. Titerswere monitored as above and mice are euthanized at the end of 4 monthsof injections. Histochemistry, Aβ levels, and toxicology analysis wasdetermined post mortem.

A. Materials and Methods

1. Preparation of Immunogens

Preparation of coupled Aβ peptides: four human Aβ peptide conjugates(amino acid residues 1-5, 1-12, 13-28, and 33-42, each conjugated tosheep anti-mouse IgG) were prepared by coupling through an artificialcysteine added to the Aβ peptide using the crosslinking reagentsulfo-EMCS. The Aβ peptide derivatives were synthesized with thefollowing final amino acid sequences. In each case, the location of theinserted cysteine residue is indicated by underlining. The Aβ13-28peptide derivative also had two glycine residues added prior to thecarboxyl terminal cysteine as indicated. Aβ1-12 peptideNH₂-DAEFRHDSGYEVC-COOH (SEQ ID NO:2) Aβ1-5 peptide NH₂-DAEFRC-COOH (SEQID NO:3) Aβ33-42 peptide NH₂-C-amino-heptanoic acid-GLMVGGVVIA-COOH (SEQID NO:4) Aβ13-28 peptide Ac-NH-HHQKLVFFAEDVGSNKGGC-COOH (SEQ ID NO:5)

To prepare for the coupling reaction, ten mg of sheep anti-mouse IgG(Jackson ImmunoResearch Laboratories) was dialyzed overnight against 10mM sodium borate buffer, pH 8.5. The dialyzed antibody was thenconcentrated to a volume of 2 mL using an Amicon Centriprep tube. Ten mgsulfo-EMCS

[N(γ-maleimidocuproyloxy)succinimide] (Molecular Sciences Co.) wasdissolved in one mL deionized water. A 40-fold molar excess ofsulfo-EMCS was added dropwise with stirring to the sheep anti-mouse IgGand then the solution was stirred for an additional ten min. Theactivated sheep anti-mouse IgG was purified and buffer exchanged bypassage over a 10 mL gel filtration column (Pierce Presto Column,obtained from Pierce Chemicals) equilibrated with 0.1 M NaPO4, 5 mMEDTA, pH 6.5. Antibody containing fractions, identified by absorbance at280 nm, were pooled and diluted to a concentration of approximately 1mg/mL, using 1.4 mg per OD as the extinction coefficient. A 40-foldmolar excess of Aβ peptide was dissolved in 20 mL of 10 mM NaPO4, pH8.0, with the exception of the Aβ33-42 peptide for which 10 mg was firstdissolved in 0.5 mL of DMSO and then diluted to 20 mL with the 10 mMNaPO4 buffer. The peptide solutions were each added to 10 mL ofactivated sheep anti-mouse IgG and rocked at room temperature for 4 hr.The resulting conjugates were concentrated to a final volume of lessthan 10 mL using an Amicon Centriprep tube and then dialyzed against PBSto buffer exchange the buffer and remove free peptide. The conjugateswere passed through 0.22 μm-pore size filters for sterilization and thenaliquoted into fractions of 1 mg and stored frozen at −20° C. Theconcentrations of the conjugates were determined using the BCA proteinassay (Pierce Chemicals) with horse IgG for the standard curve.Conjugation was documented by the molecular weight increase of theconjugated peptides relative to that of the activated sheep anti-mouseIgG. The Aβ 1-5 sheep anti-mouse conjugate was a pool of twoconjugations, the rest were from a single preparation.

2. Preparation of Aggregated Aβ Peptides

Human 1-40 (AN1528; California Peptides Inc., Lot ME0541), human 1-42(AN1792; California Peptides Inc., Lots ME0339 and ME0439), human 25-35,and rodent 1-42 (California Peptides Inc., Lot ME0218) peptides werefreshly solubilized for the preparation of each set of injections fromlyophilized powders that had been stored desiccated at −20° C. For thispurpose, two mg of peptide were added to 0.9 ml of deionized water andthe mixture was vortexed to generate a relatively uniform solution orsuspension. Of the four, AN1528 was the only peptide soluble at thisstep. A 100 μl aliquot of 10×PBS (1×PBS: 0.15 M NaCl, 0.01 M sodiumphosphate, pH 7.5) was then added at which point AN1528 began toprecipitate. The suspension was vortexed again and incubated overnightat 37° C. for use the next day.

Preparation of the pBx6 protein: An expression plasmid encoding pBx6, afusion protein consisting of the 100-amino acid bacteriophage MS-2polymerase N-terminal leader sequence followed by amino acids 592-695 ofAPP (βAPP) was constructed as described by Oltersdorf et al., J. Biol.Chem. 265, 4492-4497 (1990). The plasmid was transfected into E. coliand the protein was expressed after induction of the promoter. Thebacteria were lysed in 8M urea and pBx6 was partially purified bypreparative SDS PAGE. Fractions containing pBx6 were identified byWestern blot using a rabbit anti-pBx6 polyclonal antibody, pooled,concentrated using an Amicon Centriprep tube and dialysed against PBS.The purity of the preparation, estimated by Coomassie Blue stained SDSPAGE, was approximately 5 to 10%.

B. Results and Discussion

1. Study Design

One hundred male and female, nine- to eleven-month old heterozygousPDAPP transgenic mice were obtained from Charles River Laboratory andTaconic Laboratory. The mice were sorted into ten groups to be immunizedwith different regions of Aβ or APP combined with Freund's adjuvant.Animals were distributed to match the gender, age, parentage and sourceof the animals within the groups as closely as possible. The immunogensincluded four Aβ peptides derived from the human sequence, 1-5, 1-12,13-28, and 33-42, each conjugated to sheep anti-mouse IgG; fouraggregated Aβ peptides, human 1-40 (AN1528), human 1-42 (AN1792), human25-35, and rodent 1-42; and a fusion polypeptide, designated as pBx6,containing APP amino acid residues 592-695. A tenth group was immunizedwith PBS combined with adjuvant as a control.

For each immunization, 100 μg of each Aβ peptide in 200 μl PBS or 200 μgof the APP derivative pBx6 in the same volume of PBS or PBS alone wasemulsified 1:1 (vol:vol) with Complete Freund's adjuvant (CFA) in afinal volume of 400 μl for the first immunization, followed by a boostof the same amount of immunogen in Incomplete Freund's adjuvant (IFA)for the subsequent four doses and with PBS for the final dose.Immunizations were delivered intraperitoneally on a biweekly schedulefor the first three doses, then on a monthly schedule thereafter.Animals were bled four to seven days following each immunizationstarting after the second dose for the measurement of antibody titers.Animals were euthanized approximately one week after the final dose.

2. Aβ and APP Levels in the Brain

Following about four months of immunization with the various Aβ peptidesor the APP derivative, brains were removed from saline-perfused animals.One hemisphere was prepared for immunohistochemical analysis and thesecond was used for the quantitation of Aβ and APP levels. To measurethe concentrations of various forms of beta amyloid peptide and amyloidprecursor protein, the hemisphere was dissected and homogenates of thehippocampal, cortical, and cerebellar regions were prepared in 5 Mguanidine. These were diluted and the level of amyloid or APP wasquantitated by comparison to a series of dilutions of standards of Aβpeptide or APP of known concentrations in an ELISA format.

The median concentration of total Aβ for the control group immunizedwith PBS was 5.8-fold higher in the hippocampus than in the cortex(median of 24,318 ng/g hippocampal tissue compared to 4,221 ng/g for thecortex). The median level in the cerebellum of the control group (23.4ng/g tissue) was about 1,000-fold lower than in the hippocampus. Theselevels are similar to those that we have previously reported forheterozygous PDAPP transgenic mice of this age (Johnson-Woods et al.,1997, supra).

For the cortex, a subset of treatment groups had median total Aβ andAβ1-42 levels which differed significantly from those of the controlgroup (p<0.05), those animals receiving AN1792, rodent Aβ1-42 or theAβ1-5 peptide conjugate as shown in FIG. 11. The median levels of totalAβ were reduced by 75%, 79% and 61%, respectively, compared to thecontrol for these treatment groups. There were no discemablecorrelations between Aβ-specific antibody titers and Aβ levels in thecortical region of the brain for any of the groups.

In the hippocampus, the median reduction of total Aβ associated withAN1792 treatment (46%, p=0.0543) was not as great as that observed inthe cortex (75%, p=0.0021). However, the magnitude of the reduction wasfar greater in the hippocampus than in the cortex, a net reduction of11,186 ng/g tissue in the hippocampus versus 3,171 ng/g tissue in thecortex. For groups of animals receiving rodent Aβ1-42 or Aβ1-5, themedian total Aβ levels were reduced by 36% and 26%, respectively.However, given the small group sizes and the high variability of theamyloid peptide levels from animal to animal within both groups, thesereductions were not significant. When the levels of Aβ1-42 were measuredin the hippocampus, none of the treatment-induced reductions reachedsignificance. Thus, due to the smaller Aβ burden in the cortex, changesin this region are a more sensitive indicator of treatment effects. Thechanges in Aβ levels measured by ELISA in the cortex are similar, butnot identical, to the results from the immunohistochemical analysis (seebelow).

Total Aβ was also measured in the cerebellum, a region typicallyminimally affected with AD pathology. None of the median Aβconcentrations of any of the groups immunized with the various Aβpeptides or the APP derivative differed from that of the control groupin this region of the brain. This result suggests that non-pathologicallevels of Aβ are unaffected by treatment.

APP concentration was also determined by ELISA in the cortex andcerebellum from treated and control mice. Two different APP assays wereutilized. The first, designated APP-á/FL, recognizes both APP-alpha (á,the secreted form of APP which has been cleaved within the Aβ sequence),and full-length forms (FL) of APP, while the second recognizes onlyAPP-α. In contrast to the treatment-associated diminution of Aβ in asubset of treatment groups, the levels of APP were unchanged in all ofthe treated compared to the control animals. These results indicate thatthe immunizations with Aβ peptides are not depleting APP; rather thetreatment effect is specific to Aβ.

In summary, total Aβ and Aβ1-42 levels were significantly reduced in thecortex by treatment with AN1792, rodent Aβ1-42 or Aβ1-5 conjugate. Inthe hippocampus, total Aβ was significantly reduced only by AN1792treatment. No other treatment-associated changes in Aβ or APP levels inthe hippocampal, cortical or cerebellar regions were significant.

2. Histochemical Analyses

Brains from a subset of six groups were prepared for immunohistochemicalanalysis, three groups immunized with the Aβ peptide conjugates Aβ1-5,Aβ1-12, and Aβ13-28; two groups immunized with the full length Aβaggregates AN1792 and AN1528 and the PBS-treated control group. Theresults of image analyses of the amyloid burden in brain sections fromthese groups are shown in FIG. 12. There were significant reductions ofamyloid burden in the cortical regions of three of the treatment groupsversus control animals. The greatest reduction of amyloid burden wasobserved in the group receiving AN1792 where the mean value was reducedby 97% (p=0.001). Significant reductions were also observed for thoseanimals treated with AN1528 (95%, p=0.005) and the Aβ1-5 peptideconjugate (67%, p=0.02).

The results obtained by quantitation of total Aβ or Aβ1-42 by ELISA andamyloid burden by image analysis differ to some extent. Treatment withAN1528 had a significant impact on the level of cortical amyloid burdenwhen measured by quantitative image analysis but not on theconcentration of total Aβ in the same region when measured by ELISA. Thedifference between these two results is likely to be due to thespecificities of the assays. Image analysis measures only insoluble Aβaggregated into plaques. In contrast, the ELISA measures all forms ofAβ, both soluble and insoluble, monomeric and aggregated. Since thedisease pathology is thought to be associated with the insolubleplaque-associated form of Aβ, the image analysis technique may have moresensitivity to reveal treatment effects. However since the ELISA is amore rapid and easier assay, it is very useful for screening purposes.Moreover it may reveal that the treatment-associated reduction of Aβ isgreater for plaque-associated than total Aβ.

To determine if the Aβ-specific antibodies elicited by immunization inthe treated animals reacted with deposited brain amyloid, a subset ofthe sections from the treated animals and the control mice were reactedwith an antibody specific for mouse IgG. In contrast to the PBS group,Aβ-containing plaques were coated with endogenous IgG for animalsimmunized with the Aβ peptide conjugates Aβ1-5, Aβ1-12, and Aβ13-28; andthe full length Aβ aggregates AN1792 and AN1528. Brains from animalsimmunized with the other Aβ peptides or the APP peptide pBx6 were notanalyzed by this assay.

3. Measurement of Antibody Titers

Mice were bled four to seven days following each immunization startingafter the second immunization, for a total of five bleeds. Antibodytiters were measured as Aβ1-42-binding antibody using a sandwich ELISAwith plastic multi-well plates coated with Aβ1-42. As shown in FIG. 13,peak antibody titers were elicited following the fourth dose for thosefour vaccines which elicited the highest titers of AN1792-specificantibodies: AN1792 (peak GMT: 94,647), AN1528 (peak GMT: 88,231), Aβ1-12conjugate (peak GMT: 47,216)and rodent Aβ1-42 (peak GMT: 10,766). Titersfor these groups declined somewhat following the fifth and sixth doses.For the remaining five immunogens, peak titers were reached followingthe fifth or the sixth dose and these were of much lower magnitude thanthose of the four highest titer groups: Aβ1-5 conjugate (peak GMT:2,356), pBx6 (peak GMT: 1,986), Aβ13-28 conjugate (peak GMT: 1,183),Aβ33-42 conjugate (peak GMT: 658), Aβ25-35 (peak GMT: 125). Antibodytiters were also measured against the homologous peptides using the sameELISA sandwich format for a subset of the immunogens, those groupsimmunized with Aβ1-5, Aβ13-28, Aβ25-35, Aβ33-42 or rodent Aβ1-42. Thesetiters were about the same as those measured against Aβ1-42 except forthe rodent Aβ1-42 immunogen in which case antibody titers against thehomologous immunogen were about two-fold higher. The magnitude of theAN1792-specific antibody titer of individual animals or the mean valuesof treatment groups did not correlate with efficacy measured as thereduction of Aβ in the cortex.

4. Lymphoproliferative Responses

Aβ-dependent lymphoproliferation was measured using spleen cellsharvested approximately one week following the final, sixth,immunization. Freshly harvested cells, 105 per well, were cultured for 5days in the presence of Aβ1-40 at a concentration of 5 μM forstimulation. Cells from a subset of seven of the ten groups were alsocultured in the presence of the reverse peptide, Aβ40-1. As a positivecontrol, additional cells were cultured with the T cell mitogen, PHA,and, as a negative control, cells were cultured without added peptide.

Lymphocytes from a majority of the animals proliferated in response toPHA. There were no significant responses to the Aβ40-1 reverse peptide.Cells from animals immunized with the larger aggregated Aβ peptides,AN1792, rodent Aβ1-42 and AN1528 proliferated robustly when stimulatedwith Aβ1-40 with the highest cpm in the recipients of AN1792. One animalin each of the groups immunized with Aβ1-12 conjugate, Aβ13-28 conjugateand Aβ25-35 proliferated in response to Aβ1-40. The remaining groupsreceiving Aβ1-5 conjugate, Aβ33-42 conjugate pBx6 or PBS had no animalswith an Aβ-stimulated response. These results are summarized in Table 5below. TABLE 5 Immunogen Conjugate Aβ Amino Acids Responders Aβ1-5  Yes 5-mer 0/7 Aβ1-12 Yes 12-mer 1/8 Aβ13-28  Yes 16-mer 1/9 Aβ25-35  11-mer1/9 Aβ33-42  Yes 10-mer  0/10 Aβ1-40 40-mer 5/8 Aβ1-42 42-mer 9/9 rAβ1-42    42-mer 8/8 pBx6 0/8 PBS  0-mer 0/8

These results show that AN1792 and AN1528 stimulate strong T cellresponses, most likely of the CD4+ phenotype. The absence of anAβ-specific T cell response in animals immunized with Aβ1-5 is notsurprising since peptide epitopes recognized by CD4+ T cells are usuallyabout 15 amino acids in length, although shorter peptides can sometimesfunction with less efficiency. Thus the majority of helper T cellepitopes for the four conjugate peptides are likely to reside in the IgGconjugate partner, not in the Aβ region. This hypothesis is supported bythe very low incidence of proliferative responses for animals in each ofthese treatment groups. Since the Aβ1-5 conjugate was effective atsignificantly reducing the level of Aβ in the brain, in the apparentabsence of Aβ-specific T cells, the key effector immune response inducedby immunization with this peptide appears to be antibody.

Lack of T-cell and low antibody response from fusion peptide pBx6,encompassing APP amino acids 592-695 including all of the Aβ residuesmay be due to the poor immunogenicity of this particular preparation.The poor immunogenicity of the Aβ25-35 aggregate is likely due to thepeptide being too small to be likely to contain a good T cell epitope tohelp the induction of an antibody response. If this peptide wereconjugated to a carrier protein, it would probably be more immunogenic.

V. Preparation of Polyclonal Antibodies for Passive Protection

125 non-transgenic mice were immunized with Aβ, plus adjuvant, andeuthanized at 4-5months. Blood was collected from immunized mice. IgGwas separated from other blood components. Antibody specific for theimmunogen may be partially purified by affinity chromatography. Anaverage of about 0.5-1 mg of immunogen-specific antibody is obtained permouse, giving a total of 60-120 mg.

VI. Passive Immunization with Antibodies to Aβ

Groups of 7-9 month old PDAPP mice each are injected with 0.5 mg in PBSof polyclonal anti-Aβ or specific anti-Aβ monoclonals as shown below.The cell line designated RB44-10D5.19.21 producing the antibody 10D5 hasthe American Type Culture Collection (ATCC) accession number PTA-5129,having been deposited on Apr. 8, 2003. The cell line producing theantibody 266 has the ATCC accession number ______, having been depositedon Jul. 20, 2004.

All antibody preparations are purified to have low endotoxin levels.Monoclonals can be prepared against a fragment by injecting the fragmentor longer form of Aβ into a mouse, preparing hybridomas and screeningthe hybridomas for an antibody that specifically binds to a desiredfragment of Aβ without binding to other nonoverlapping fragments of Aβ.TABLE 6 Antibody Epitope 2H3 Aβ 1-12 10D5 Aβ 1-12 266 Aβ 13-28  21F12 Aβ33-42  Mouse polyclonal Anti-Aggregated Aβ42 anti-human Aβ42

Mice were injected ip as needed over a 4 month period to maintain acirculating antibody concentration measured by ELISA titer of greaterthan 1/1000 defined by ELISA to Aβ42 other immunogen. Titers weremonitored as above and mice were euthanized at the end of 6 months ofinjections. Histochemistry, Aβ levels and toxicology were performed postmortem. Ten mice were used per group.

VII. Comparison of Different Adjuvants

This example compares CFA, alum, an oil-in water emulsion and MPL forcapacity to stimulate an immune response.

A. Materials and Methods

1. Study Design

One hundred female Hartley strain six-week old guinea pigs, obtainedfrom Elm Hill, were sorted into ten groups to be immunized with AN1792or a palmitoylated derivative therof combined with various adjuvants.Seven groups received injections of AN1792 (33 μg unless otherwisespecified) combined with a) PBS, b) Freund's adjuvant, c) MPL, d)squalene, e) MPL/squalene f) low dose alum, or g) high dose alum (300 μgAN1792). Two groups received injections of a palmitoylated derivative ofAN1792 (33 μg) combined with a) PBS or b) squalene. A final, tenth groupreceived PBS alone without antigen or additional adjuvant. For the groupreceiving Freund's adjuvant, the first dose was emulsified with CFA andthe remaining four doses with IFA. Antigen was administered at a dose of33 μg for all groups except the high dose alum group, which received 300μg of AN1792. Injections were administered intraperitoneally for CFA/IFAand intramuscularly in the hind limb quadriceps alternately on the rightand left side for all other groups. The first three doses were given ona biweekly schedule followed by two doses at a monthly interval). Bloodwas drawn six to seven days following each immunization, starting afterthe second dose, for measurement of antibody titers.

2. Preparation of Immunogens

Two mg Aβ42 (California Peptide, Lot ME0339) was added to 0.9 ml ofdeionized water and the mixture was vortexed to generate a relativelyuniform suspension. A 100 μl aliquot of 10×PBS (1×PBS, 0.15 M NaCl, 0.01M sodium phosphate, pH 7.5) was added. The suspension was vortexed againand incubated overnight at 37° C. for use the next day. Unused Aβ1-42was stored with desiccant as a lyophilized powder at −20° C.

A palmitoylated derivative of AN1792 was prepared by coupling palmiticanhydride, dissolved in dimethyl formamide, to the amino terminalresidue of AN1792 prior to removal of the nascent peptide from the resinby treatment with hydrofluoric acid.

To prepare vaccine doses with Complete Freund's adjuvant (CFA) (group2), 33 μg of AN1792 in 200 μl PBS was emulsified 1:1 (vol:vol) with CFAin a final volume of 400 μl for the first immunization. For subsequentimmunizations, the antigen was similarly emulsified with IncompleteFreund's adjuvant (IFA).

To prepare vaccine doses with MPL for groups 5 and 8, lyophilized powder(Ribi ImmunoChem Research, Inc., Hamilton, Mont.) was added to 0.2%aqueous triethylamine to a final concentration of 1 mg/ml and vortexed.The mixture was heated to 65 to 70° C. for 30 sec to create a slightlyopaque uniform suspension of micelles. The solution was freshly preparedfor each set of injections. For each injection in group 5, 33 μg ofAN1792 in 16.5 μl PBS, 50 μg of MPL (50 μl) and 162 μl of PBS were mixedin a borosilicate tube immediately before use.

To prepare vaccine doses with the low oil-in-water emulsion, AN1792 inPBS was added to 5% squalene, 0.5% Tween 80, 0.5% Span 85 in PBS toreach a final single dose concentration of 33 μg AN1792 in 250 μl (group6). The mixture was emulsified by passing through a two-chamberedhand-held device 15 to 20 times until the emulsion droplets appeared tobe about equal in diameter to a 1.0 μm diameter standard latex bead whenviewed under a microscope. The resulting suspension was opalescent,milky white. The emulsions were freshly prepared for each series ofinjections; For group 8, MPL in 0.2% triethylamine was added at aconcentration of 50 μg per dose to the squalene and detergent mixturefor emulsification as noted above. For the palmitoyl derivative (group7), 33 μg per dose of palmitoyl-NH-Aβ1-42 was added to squalene andvortexed. Tween 80 and Span 85 were then added with vortexing. Thismixture was added to PBS to reach final concentrations of 5% squalene,0.5% Tween 80, 0.5% Span 85 and the mixture was emulsified as notedabove.

To prepare vaccine doses with alum (groups 9 and 10), AN1792 in PBS wasadded to Alhydrogel (aluminum hydroxide gel, Accurate, Westbury, N.Y.)to reach concentrations of 33μg (low dose, group 9) or 300 μg (highdose, group 10) AN1792 per 5 mg of alum in a final dose volume of 250μl. The suspension was gently mixed for 4 hr at RT.

3. Measurement of Antibody Titers

Guinea pigs were bled six to seven days following immunization startingafter the second immunization for a total of four bleeds. Antibodytiters against Aβ42 were measured by ELISA as described in GeneralMaterials and Methods.

4. Tissue Preparation

After about 14 weeks, all guinea pigs were administered CO2.Cerebrospinal fluid was collected and the brains were removed and threebrain regions (hippocampus, cortex and cerebellum) were dissected andused to measure the concentration of total Aβ protein using ELISA.

B. Results

1. Antibody Responses

There was a wide range in the potency of the various adjuvants whenmeasured as the antibody response to AN1792 following immunization. Asshown in FIG. 14, when AN1792 was administered in PBS, no antibody wasdetected following two or three immunizations and negligible responseswere detected following the fourth and fifth doses with geometric meantiters (GMTs) of only about 45. The o/w emulsion induced modest titersfollowing the third dose (GMT 255) that were maintained following thefourth dose (GMT 301) and fell with the final dose (GMT 54). There was aclear antigen dose response for AN1792 bound to alum with 300 μg beingmore immunogenic at all time points than 33 μg. At the peak of theantibody response, following the fourth immunization, the differencebetween the two doses was 43% with GMTs of about 1940 (33 μg) and 3400(300 μg). The antibody response to 33 μg AN1792 plus MPL was verysimilar to that generated with almost a ten-fold higher dose of antigen(300 μg) bound to alum. The addition of MPL to an o/w emulsion decreasedthe potency of the vaccine relative to that with MPL as the soleadjuvant by as much as 75%. A palmitoylated derivative of AN1792 wascompletely non-immunogenic when administered in PBS and gave modesttiters when presented in an o/w emulsion with GMTs of 340 and 105 forthe third and fourth bleeds. The highest antibody titers were generatedwith Freund's adjuvant with a peak GMT of about 87,000, a value almost30-fold greater than the GMTs of the next two most potent vaccines, MPLand high dose AN1792/alum.

The most promising adjuvants identified in this study are MPL and alum.Of these two, MPL appears preferable because a 10-fold lower antigendose was required to generate the same antibody response as obtainedwith alum. The response can be increased by increasing the dose ofantigen and/or adjuvant and by optimizing the immunization schedule. Theo/w emulsion was a very weak adjuvant for AN1792 and adding an o/wemulsion to MPL adjuvant diminished the intrinsic adjuvant activity ofMPL alone.

2. Aβ Levels in the Brain

At about 14 weeks the guinea pigs were deeply anesthetized, thecerebrospinal fluid (CSF) was drawn and brains were excised from animalsin a subset of the groups, those immunized with Freund's adjuvant (group2), MPL (group 5), alum with a high dose, 300 μg, of AN1792 (group 10)and the PBS immunized control group (group 3). To measure the level ofAβ peptide, one hemisphere was dissected and homogenates of thehippocampal, cortical, and cerebellar regions were prepared in 5 Mguanidine. These were diluted and quantitated by comparison to a seriesof dilutions of Aβ standard protein of known concentrations in an ELISAformat. The levels of Aβ protein in the hippocampus, the cortex and thecerebellum were very similar for all four groups despite the wide rangeof antibody responses to Aβ elicited by these vaccines. Mean Aβ levelsof about 25 ng/g tissue were measured in the hippocampus, 21 ng/g in thecortex, and 12 ng/g in the cerebellum. Thus, the presence of a highcirculating antibody titer to Aβ for almost three months in some ofthese animals did not alter the total Aβ levels in their brains. Thelevels of Aβ in the CSF were also quite similar between the groups. Thelack of large effect of AN1792 immunization on endogenous Aβ indicatesthat the immune response is focused on pathological formations of Aβ.

VIII. Immune Response to Different Adjuvants in Mice

Six-week old female Swiss Webster mice were used for this study with10-13 animals per group. Immunizations were given on days 0, 14, 28, 60,90 and 20 administered subcutaneously in a dose volume of 200 μl. PBSwas used as the buffer for all formulations. Animals were bleed sevendays following each immunization starting after the second dose foranalysis of antibody titers by ELISA. The treatment regime of each groupis summarized in Table 7. TABLE 7 Experimental Design of Study 010 GroupN^(a) Adjuvant^(b) Dose Antigen Dose(μg) 1 10 MPL 12.5 μg AN1792 33 2 10MPL 25 μg AN1792 33 3 10 MPL 50 μg AN1792 33 4 13 MPL 125 μg AN1792 33 513 MPL 50 μg AN1792 150 6 13 MPL 50 μg AN1528 33 7 10 PBS AN1792 33 8 10PBS None 9 10 Squalene 5% AN1792 33 emulsified 10 10 Squalene 5% AN179233 admixed 11 10 Alum 2 mg AN1792 33 12 13 MPL + Alum 50 μg/2 mg AN179233 13 10 QS-21 5 μg AN1792 33 14 10 QS-21 10 μg AN1792 33 15 10 QS-21 25AN1792 AN1792 33 16 13 QS-21 25 AN1792 AN1792 150 17 13 QS-21 25 AN1792AN1528 33 18 13 QS-21 + MPL 25 μg/50 μg AN1792 33 19 13 QS-21 + 25 μg/2mg AN1792 33 AlumFootnotes:^(a)Number of mice in each group at the initiation of the experiment.^(b)The adjuvants are noted. The buffer for all these formulations wasPBS. For group 8, there was no adjuvant and no antigen.

The ELISA titers of antibodies against Aβ42 in each group are shown inTable 8 below. TABLE 8 Geometric Mean Antibody Titers Treatment Week ofBleed Group 2.9 5.0 8.7 12.9 16.7 1 248 1797 2577 6180 4177 2 598 31143984 5287 6878 3 1372 5000 7159 12333 12781 4 1278 20791 14368 2009725631 5 3288 26242 13229 9315 23742 6 61 2536 2301 1442 4504 7 37 395484 972 2149 8 25 25 25 25 25 9 25 183 744 952 1823 10 25 89 311 513 81711 29 708 2618 2165 3666 12 198 1458 1079 612 797 13 38 433 566 1080 62614 104 541 3247 1609 838 15 212 2630 2472 1224 1496 16 183 2616 66802085 1631 17 28 201 375 222 1540 18 31699 15544 23095 6412 9059 19 63243 554 299 441The table shows that the highest titers were obtained for groups 4, 5and 18, in which the adjuvants were 125 μg MPL, 50 μg MPL and QS-21 plusMPL.

IX. Therapeutic Efficacy of Different Adjuvants

A therapeutic efficacy study was conducted in PDAPP transgenic mice witha set of adjuvants suitable for use in humans to determine their abilityto potentiate immune responses to Aβ and to induce the immune-mediatedclearance of amyloid deposits in the brain.

One hundred eighty male and female, 7.5- to 8.5-month old heterozygousPDAPP transgenic mice were obtained from Charles River Laboratories. Themice were sorted into nine groups containing 15 to 23 animals per groupto be immunized with AN1792 or AN1528 combined with various adjuvants.Animals were distributed to match the gender, age, and parentage of theanimals within the groups as closely as possible. The adjuvants includedalum, MPL, and QS-21, each combined with both antigens, and Freund'sadjuvant (FA) combined with only AN1792. An additional group wasimmunized with AN1792 formulated in PBS buffer plus the preservativethimerosal without adjuvant. A ninth group was immunized with PBS aloneas a negative control.

Preparation of aggregated Aβ peptides: human Aβ1-40 (AN1528; CaliforniaPeptides Inc., Napa, Calif.; Lot ME0541) and human Aβ1-42 (AN1792;California Peptides Inc., Lot ME0439) peptides were freshly solubilizedfor the preparation of each set of injections from lyophilized powdersthat had been stored desiccated at −20° C. For this purpose, two mg ofpeptide were added to 0.9 ml of deionized water and the mixture wasvortexed to generate a relatively uniform solution or suspension. AN1528was soluble at this step, in contrast to AN1792. A 100 μl aliquot of10×PBS (1×PBS: 0.15 M NaCl, 0.01 M sodium phosphate, pH 7.5) was thenadded at which point AN1528 began to precipitate. The suspensions werevortexed again and incubated overnight at 37° C. for use the next day.

To prepare vaccine doses with alum, Aβ peptide in PBS was added toAlhydrogel (two percent aqueous aluminum hydroxide gel, Sargeant, Inc.,Clifton, N.J.) to reach concentrations of 100 μg Aβ peptide per 2 mg ofalum. 10×PBS was added to a final dose volume of 200 μl in 1×PBS. Thesuspension was then gently mixed for approximately 4 hr at RT prior toinjection.

To prepare vaccine doses for with MPL (Groups 2 and 6), lyophilizedpowder (Ribi ImmunoChem Research, Inc., Hamilton, Mont.; Lot67039-E0896B) was added to 0.2% aqueous triethylamine to a finalconcentration of 1 mg/ml and vortexed. The mixture was heated to 65 to70° C. for 30 sec to create a slightly opaque uniform suspension ofmicelles. The solution was stored at 4° C. For each set of injections,100 μg of peptide per dose in 50 μl PBS, 50 μg of MPL per dose (50 μl)and 100 μl of PBS per dose were mixed in a borosilicate tube immediatelybefore use.

To prepare vaccine doses with QS-21 (Groups 3 and 7), lyophilized powder(Aquila, Framingham, Mass.; Lot A7018R) was added to PBS, pH 6.6-6.7 toa final concentration of 1 mg/ml and vortexed. The solution was storedat −20° C. For each set of injections, 100 μg of peptide per dose in 50μl PBS, 25 μg of QS-21 per dose in 25 μl PBS and 125 μl of PBS per dosewere mixed in a borosilicate tube immediately before use.

To prepare vaccine doses with Freund's Adjuvant (Group 4), 100 μg ofAN1792 in 200 μl PBS was emulsified 1:1 (vol:vol) with Complete Freund'sAdjuvant (CFA) in a final volume of 400 μl for the first immunization.For subsequent immunizations, the antigen was similarly emulsified withIncomplete Freund's Adjuvant (IFA). For the vaccines containing theadjuvants alum, MPL or QS-21, 100 μg per dose of AN1792 or AN1528 wascombined with alum (2 mg per dose) or MPL (50 μg per dose) or QS-21 (25μg per dose) in a final volume of 200 μl PBS and delivered bysubcutaneous inoculation on the back between the shoulder blades. Forthe group receiving FA, 100 μg of AN1792 was emulsified 1:1 (vol:vol)with Complete Freund's adjuvant (CFA) in a final volume of 400 μl anddelivered intraperitoneally for the first immunization, followed by aboost of the same amount of immunogen in Incomplete Freund's adjuvant(IFA) for the subsequent five doses. For the group receiving AN1792without adjuvant, 10μg AN1792 was combined with 5 μg thimerosal in afinal volume of 50 μl PBS and delivered subcutaneously. The ninth,control group received only 200 μl PBS delivered subcutaneously.Immunizations were given on a biweekly schedule for the first threedoses, then on a monthly schedule thereafter on days 0, 16, 28, 56, 85and 112. Animals were bled six to seven days following each immunizationstarting after the second dose for the measurement of antibody titers.Animals were euthanized approximately one week after the final dose.Outcomes were measured by ELISA assay of Aβ and APP levels in brain andby immunohistochemical evaluation of the presence of amyloid plaques inbrain sections. In addition, Aβ-specific antibody titers, andAβ-dependent proliferative and cytokine responses were determined.

Table 9 shows that the highest antibody titers to Aβ1-42 were elicitedwith FA and AN1792, titers which peaked following the fourthimmunization (peak GMT: 75,386) and then declined by 59% after thefinal, sixth immunization. The peak, mean titer elicited by MPL withAN1792 was 62% lower than that generated with FA (peak GMT: 28,867) andwas also reached early in the immunization scheme, after 3 doses,followed by a decline to 28% of the peak value after the sixthimmunization. The peak mean titer generated with QS-21 combined withAN1792 (GMT: 1,511) was about 5-fold lower than obtained with MPL. Inaddition, the kinetics of the response were slower, since an additionalimmunization was required to reach the peak response. Titers generatedby alum-bound AN1792 were marginally greater than those obtained withQS-21 and the response kinetics were more rapid. For AN1792 delivered inPBS with thimerosal the frequency and size of titers were barely greaterthan that for PBS alone. The peak titers generated with MPL and AN1528(peak GMT 3099) were about 9-fold lower than those with AN1792.Alum-bound AN1528 was very poorly immunogenic with low titers generatedin only some of the animals. No antibody responses were observed in thecontrol animals immunized with PBS alone. TABLE 9 Geometric MeanAntibody Titers^(a) Week of Bleed Treatment 3.3 5.0 9.0 13.0 17.0 Alum/102 1,081 2,366 1,083 572 AN1792 (12/21)^(b) (17/20) (21/21) (19/21)(18/21) MPL/ 6241 28,867 1,1242 5,665 8,204 AN1792 (21/21) (21/21)(21/21) (20/20) (20/20) QS-21/ 30 227 327 1,511 1,188 AN1792 (1/20)(10/19) (10/19) (17/18) (14/18) CFA/ 10,076 61,279 75,386 41,628 30,574AN1792 (15/15) (15/15) (15/15) (15/15) (15/15) Alum/ 25 33 39 37 31AN1528 (0/21) (1/21) (3/20) (1/20) (2/20) MPL/ 184 2,591 1,653 1,1563,099 AN1528 (15/21) (20/21) (21/21) (20/20) (20/20) QS-21/ 29 221 51820 2,994 AN1528 (1/22) (13/22) (4/22) (20/22) (21/22) PBS plus 25 33 3937 47 Thimerosal (0/16) (2/16) (4/16) (3/16) (4/16) PBS 25 25 25 25 25(0/16) (0/16) (0/15) (0/12) (0/16)Footnotes:^(a)Geometric mean antibody titers measured against Aβ1-42^(b)Number of responders per group

The results of AN1792 or AN1592 treatment with various adjuvants, orthimerosal on cortical amyloid burden in 12-month old mice determined byELISA are shown in FIGS. 15A-15E. In PBS control PDAPP mice (FIG. 15A),the median level of total A in the cortex at 12 months was 1,817 ng/g.Notably reduced levels of A were observed in mice treated with AN1792plus CFA/IFA (FIG. 15C), AN1792 plus alum (FIG. 15D), AN1792 plus MPL(FIG. 15E) and QS21 plus AN1792 (FIG. 15E). The reduction reachedstatistical significance (p<0.05) only for AN1792 plus CFA/IFA (FIG.15C). However, as shown in Examples I and III, the effects ofimmunization in reducing A levels become substantially greater in 15month and 18 month old mice. Thus, it is expected that at least theAN1792 plus alum, AN1792 plus MPL and AN1792 plus QS21 compositions willachieve statistical significance in treatment of older mice. Bycontrast, the AN1792 plus the preservative thimerosal (FIG. 15D) showeda median level of A about the same as that in the PBS treated mice.Similar results were obtained when cortical levels of A42 were compared.The median level of A42 in PBS controls was 1624 ng/g. Notably reducedmedian levels of 403, 1149, 620 and 714 were observed in the micetreated with AN1792 plus CFA/IFA, AN1792 plus alum, AN1792 plus MPL andAN1792 plus QS21 respectively, with the reduction achieving statisticalsignificance (p=0.05) for the AN1792 CFA/IFA treatment group. The medianlevel in the AN1792 thimerosal treated mice was 1619 ng/g A42.

X. Toxicity Analysis

Tissues were collected for histopathologic examination at thetermination of studies described in Examples 2, 3 and 7. In addition,hematology and clinical chemistry were performed on terminal bloodsamples from Examples 3 and 7. Most of the major organs were evaluated,including brain, pulmonary, lymphoid, gastrointestinal, liver, kidney,adrenal and gonads. Although sporadic lesions were observed in the studyanimals, there were no obvious differences, either in tissues affectedor lesion severity, between AN1792 treated and untreated animals. Therewere no unique histopathological lesions noted in AN-1528-immunizedanimals compared to PBS-treated or untreated animals. There were also nodifferences in the clinical chemistry profile between adjuvant groupsand the PBS treated animals in Example 7. Although there weresignificant increases in several of the hematology parameters betweenanimals treated with AN1792 and Freund's adjuvant in Example 7 relativeto PBS treated animals, these type of effects are expected from Freund'sadjuvant treatment and the accompanying peritonitis and do not indicateany adverse effects from AN1792 treatment. Although not part of thetoxicological evaluation, PDAPP mouse brain pathology was extensivelyexamined as part of the efficacy endpoints. No sign of treatment relatedadverse effect on brain morphology was noted in any of the studies.These results indicate that AN1792 treatment is well tolerated and atleast substantially free of side effects.

XI. Therapeutic Treatment with Anti-Aβ Antibodies

This examples tests the capacity of various monoclonal and polyclonalantibodies to Aβ to inhibit accumulation of Aβ in the brain ofheterozygotic transgenic mice.

1. Study Design

Sixty male and female, heterozygous PDAPP transgenic mice, 8.5 to 10.5months of age were obtained from Charles River Laboratory. The mice weresorted into six groups to be treated with various antibodies directed toAβ. Animals were distributed to match the gender, age, parentage andsource of the animals within the groups as closely as possible. As shownin Table 10, the antibodies included four murine Aβ-specific monoclonalantibodies, 2H3 (directed to Aβ residues 1-12), 10D5 (directed to Aβresidues 1-16) (details of the deposit of 10D5 are discussed in ExampleVI, supra), 266 (directed to Aβ residues 13-28 and binds to monomericbut not to aggregated AN1 792) (details of the deposit of 266 arediscussed in Example VI, supra), 21F12 (directed to A residues 33-42). Afifth group was treated with an Aβ-specific polyclonal antibody fraction(raised by immunization with aggregated AN1792). The negative controlgroup received the diluent, PBS, alone without antibody.

The monoclonal antibodies were injected at a dose of about 10 mg/kg(assuming that the mice weighed 50 g). Injections were administeredintraperitoneally every seven days on average to maintain anti-Aβ titersabove 1000. Although lower titers were measured for mAb 266 since itdoes not bind well to the aggregated AN1792 used as the capture antigenin the assay, the same dosing schedule was maintained for this group.The group receiving monoclonal antibody 2H3 was discontinued within thefirst three weeks since the antibody was cleared too rapidly in vivo.Animals were bled prior to each dosing for the measurement of antibodytiters. Treatment was continued over a six-month period for a total of196 days. Animals were euthanized one week after the final dose. TABLE10 EXPERIMENTAL DESIGN OF STUDY 006 Treatment Treatment AntibodyAntibody Group N^(a) Antibody Specificity Isotype 1 9 none NA^(b) NA(PBS alone) 2 10 Polyclonal Aβ1-42 mixed 3 0 mAb^(c) 2H3 Aβ1-12 IgG1 4 8mAb 10D5 Aβ1-16 IgG1 5 6 mAb 266 Aβ13-28 IgG1 6 8 mAb 21F12 Aβ33-42IgG2aFootnotes^(a)Number of mice in group at termination of the experiment. All groupsstarted with 10 animals per group.^(b)NA: not applicable^(c)mAb: monoclonal antibody

2. Materials and Methods

a. Preparation of the Antibodies

The anti-Aβ polyclonal antibody was prepared from blood collected fromtwo groups of animals. The first group consisted of 100 female SwissWebster mice, 6 to 8 weeks of age. They were immunized on days 0, 15,and 29 with 100 μg of AN1792 combined with CFA/IFA. A fourth injectionwas given on day 36 with one-half the dose of AN1792. Animals wereexsanguinated upon sacrifice at day 42, serum was prepared and the serawere pooled to create a total of 64 ml. The second group consisted of 24female mice isogenic with the PDAPP mice but nontransgenic for the humanAPP gene, 6 to 9 weeks of age. They were immunized on days 0, 14, 28 and56 with 100 μg of AN1792 combined with CFA/IFA. These animals were alsoexsanguinated upon sacrifice at day 63, serum was prepared and pooledfor a total of 14 ml. The two lots of sera were pooled. The antibodyfraction was purified using two sequential rounds of precipitation with50% saturated ammonium sulfate. The final precipitate was dialyzedagainst PBS and tested for endotoxin. The level of endotoxin was lessthan 1 EU/mg.

The anti-Aβ monoclonal antibodies were prepared from ascities fluid. Thefluid was first delipidated by the addition of concentrated sodiumdextran sulfate to ice-cold ascites fluid by stirring on ice to a reacha final concentration of 0.238%. Concentrated CaCl₂ was then added withstirring to reach a final concentration of 64 mM. This solution wascentrifuged at 10,000×g and the pellet was discarded. The supernatantwas stirred on ice with an equal volume of saturated ammonium sulfateadded dropwise. The solution was centrifuged again at 10,000×g and thesupernatant was discarded. The pellet was resuspended and dialyzedagainst 20 mM Tris-HCl, 0.4 M NaCl, pH 7.5. This fraction was applied toa Pharmacia FPLC Sepharose Q Column and eluted with a reverse gradientfrom 0.4 M to 0.275 M NaCl in 20 mM Tris-HCl, pH 7.5.

The antibody peak was identified by absorbance at 280 nm and appropriatefractions were pooled. The purified antibody preparation wascharacterized by measuring the protein concentration using the BCAmethod and the purity using SDS-PAGE. The pool was also tested forendotoxin. The level of endotoxin was less than 1 EU/mg. titers, titersless than 100 were arbitrarily assigned a titer value of 25.

3. Aβ and APP Levels in the Brain:

Following about six months of treatment with the various anti-Aβantibody preparations, brains were removed from the animals followingsaline perfusion. One hemisphere was prepared for immunohistochemicalanalysis and the second was used for the quantitation of Aβ and APPlevels. To measure the concentrations of various forms of beta amyloidpeptide and amyloid precursor protein (APP), the hemisphere wasdissected and homogenates of the hippocampal, cortical, and cerebellarregions were prepared in 5M guanidine. These were serially diluted andthe level of amyloid peptide or APP was quantitated by comparison to aseries of dilutions of standards of Aβ peptide or APP of knownconcentrations in an ELISA format.

The levels of total Aβ and of Aβ1-42 measured by ELISA in homogenates ofthe cortex, and the hippocampus and the level of total Aβ in thecerebellum are shown in Tables 11, 12, and 13, respectively. The medianconcentration of total Aβ for the control group, inoculated with PBS,was 3.6-fold higher in the hippocampus than in the cortex (median of63,389 ng/g hippocampal tissue compared to 17,818 ng/g for the cortex).The median level in the cerebellum of the control group (30.6 ng/gtissue) was more than 2,000-fold lower than in the hippocampus. Theselevels are similar to those that we have previously reported forheterozygous PDAPP transgenic mice of this age (Johnson-Woods et al.,1997).

For the cortex, one treatment group had a median Aβ level, measured asAβ1-42, which differed significantly from that of the control group(p<0.05), those animals receiving the polyclonal anti-Aβ antibody asshown in Table 11. The median level of Aβ1-42 was reduced by 65%,compared to the control for this treatment group. The median levels ofAβ1-42 were also significantly reduced by 55% compared to the control inone additional treatment group, those animals dosed with the mAb 10D5(p=0.0433). TABLE 11 CORTEX Medians Means Treatment Total Aβ Aβ42 TotalAβ Aβ42 Group N^(a) ELISA value^(b) P value^(c) % Change ELISA value Pvalue % Change ELISA value ELISA value PBS 9 17818 NA^(d) NA 13802 NA NA16150 +/− 7456^(e)   12621 +/− 5738  Polyclonal 10 6160 0.0055 −65 48920.0071 −65 5912 +/− 4492 4454 +/− 3347 anti-Aβ42 mAb 10D5 8 7915 0.1019−56 6214 0.0433 −55 9695 +/− 6929 6943 +/− 3351 mAb 266 6 9144 0.1255−49 8481 0.1255 −39 9204 +/− 9293 7489 +/− 6921 mAb 21F12 8 15158 0.2898−15 13578 0.7003 −2 12481 +/− 7082  11005 +/− 6324 Footnotes:^(f)Number of animals per group at the end of the experiment^(g)ng/g tissue^(h)Mann Whitney analysis^(i)NA: not applicable^(j)Standard Deviation

In the hippocampus, the median percent reduction of total Aβ associatedwith treatment with polyclonal anti-Aβ antibody (50%, p=0.0055) was notas great as that observed in the cortex (65%) (Table 12). However, theabsolute magnitude of the reduction was almost 3-fold greater in thehippocampus than in the cortex, a net reduction of 31,683 ng/g tissue inthe hippocampus versus 11,658 ng/g tissue in the cortex. When measuredas the level of the more amyloidogenic form of Aβ, Aβ1-42, rather thanas total Aβ, the reduction achieved with the polyclonal antibody wassignificant (p=0.0025). The median levels in groups treated with themAbs 10D5 and 266 were reduced by 33% and 21%, respectively. TABLE 12HIPPOCAMPUS Medians Total Aβ Aβ42 Means Treatment ELISA % ELISA % TotalAβ Aβ42 Group N^(a) value^(b) P value^(c) Change value P value ChangeELISA value ELISA value PBS 9 63389 NA^(d) NA 54429 NA NA   58351 +/−13308^(e) 52801 +/− 14701 Polyclonal 10 31706 0.0055 −50 27127 0.0025−50 30058 +/− 22454 24853 +/− 18262 anti-Aβ42 mAb 10D5 8 46779 0.0675−26 36290 0.0543 −33 44581 +/− 18632 36465 +/− 17146 mAb 266 6 486890.0990 −23 43034 0.0990 −21 36419 +/− 27304 32919 +/− 25372 mAb 21F12 851563 0.7728 −19 47961 0.8099 −12 57327 +/− 28927 50305 +/− 23927Footnotes:^(a)Number of animals per group at the end of the experiment^(b)ng/g tissue^(c)Mann Whitney analysis^(d)NA: not applicable^(e)Standard Deviation

Total Aβ was also measured in the cerebellum (Table 13). Those groupsdosed with the polyclonal anti-Aβ and the 266 antibody showedsignificant reductions of the levels of total Aβ (43% and 46%, p=0.0033and p=0.0184, respectively) and that group treated with 10D5 had a nearsignificant reduction (29%, p=0.0675). TABLE 13 CEREBELLUM Medians MeansTotal Aβ Total Aβ Treatment ELISA P % ELISA Group N^(a) value^(b)value^(c) Change value PBS 9 30.64 NA^(d) NA    40.00 +/− 31.89^(e)Polyclonal 10 17.61 0.0033 −43 18.15 +/− 4.36 anti-Aβ42 mAb 10D5 8 21.680.0675 −29  27.29 +/− 19.43 mAb 266 6 16.59 0.0184 −46 19.59 +/− 6.59mAb 21F12 8 29.80 >0.9999 −3 32.88 +/− 9.90Footnotes:^(a)Number of animals per group at the end of the experiment^(b)ng/g tissue^(c)Mann Whitney analysis^(d)NA: not applicable^(e)Standard Deviation

APP concentration was also determined by ELISA in the cortex andcerebellum from antibody-treated and control, PBS-treated mice. Twodifferent APP assays were utilized. The first, designated APP-α/FL,recognizes both APP-alpha (α, the secreted form of APP which has beencleaved within the Aβ sequence), and full-length forms (FL) of APP,while the second recognizes only APP-α. In contrast to thetreatment-associated diminution of Aβ in a subset of treatment groups,the levels of APP were virtually unchanged in all of the treatedcompared to the control animals. These results indicate that theimmunizations with Aβ antibodies deplete Aβ without depleting APP.

In summary, Aβ levels were significantly reduced in the cortex,hippocampus and cerebellum in animals treated with the polyclonalantibody raised against AN1792. To a lesser extent monoclonal antibodiesto the amino terminal region of Aβ1-42, specifically amino acids 1-16and 13-28 also showed significant treatment effects.

4. Histochemical Analyses:

The morphology of Aβ-immunoreactive plaques in subsets of brains frommice in the PBS, polyclonal Aβ42, 21F12, 266 and 10D5 treatment groupswas qualitatively compared to that of previous studies in which standardimmunization procedures with Aβ42 were followed.

The largest alteration in both the extent and appearance of amyloidplaques occurred in the animals immunized with the polyclonal Aβ42antibody. The reduction of amyloid load, eroded plaque morphology andcell-associated Aβ immunoreactivity closely resembled effects producedby the standard immunization procedure. These observations support theELISA results in which significant reductions in both total Aβ and Aβ42were achieved by administration of the polyclonal Aβ42 antibody.

In similar qualitative evaluations, amyloid plaques in the 10D5 groupwere also reduced in number and appearance, with some evidence ofcell-associated Aβ immunoreactivity. Major differences were not seenwhen the 21F12 and 266 groups were compared with the PBS controls.

5. Measurement of Antibody Titers:

A subset of three randomly chosen mice from each group were bled justprior to each intraperitoneal inoculation, for a total of 30 bleeds.Antibody titers were measured as Aβ1-42-binding antibody using asandwich ELISA with plastic multi-well plates coated with Aβ1-42 asdescribed in detail in the General Materials and Methods. Mean titersfor each bleed are shown in FIGS. 16-18 for the polyclonal antibody andthe monoclonals 10D5 and 21F12, respectively. Titers averaged about 1000over this time period for the polyclonal antibody preparation and wereslightly above this level for the 10D5- and 21F12-treated animals.

6. Lymphoproliferative Responses:

Aβ-dependent lymphoproliferation was measured using spleen cellsharvested eight days following the final antibody infusion. Freshlyharvested cells, 10⁵ per well, were cultured for 5 days in the presenceof Aβ1-40 at a concentration of 5 μM for stimulation. As a positivecontrol, additional cells were cultured with the T cell mitogen, PHA,and, as a negative control, cells were cultured without added peptide.

Splenocytes from aged PDAPP mice passively immunized with variousanti-Aβ antibodies were stimulated in vitro with AN1792 andproliferative and cytokine responses were measured. The purpose of theseassays was to determine if passive immunization facilitated antigenpresentation, and thus priming of T cell responses specific for AN1792.No AN1792-specific proliferative or cytokine responses were observed inmice passively immunized with the anti-Aβ antibodies.

XII. Prevention and Treatment of Subjects

A single-dose phase I trial is performed to determine safety. Atherapeutic agent is administered in increasing dosages to differentpatients starting from about 0.01 the level of presumed efficacy, andincreasing by a factor of three until a level of about 10 times theeffective mouse dosage is reached.

A phase II trial is performed to determine therapeutic efficacy.Patients with early to mid Alzheimer's Disease defined using Alzheimer'sdisease and Related Disorders Association (ADRDA) criteria for probableAD are selected. Suitable patients score in the 12-26 range on theMini-Mental State Exam (MMSE). Other selection criteria are thatpatients are likely to survive the duration of the study and lackcomplicating issues such as use of concomitant medications that mayinterfere. Baseline evaluations of patient function are made usingclassic psychometric measures, such as the MMSE, and the ADAS, which isa comprehensive scale for evaluating patients with Alzheimer's Diseasestatus and function. These psychometric scales provide a measure ofprogression of the Alzheimer's condition. Suitable qualitative lifescales can also be used to monitor treatment. Disease progression canalso be monitored by MRI. Blood profiles of patients can also bemonitored including assays of immunogen-specific antibodies and T-cellsresponses.

Following baseline measures, patients begin receiving treatment. Theyare randomized and treated with either therapeutic agent or placebo in ablinded fashion. Patients are monitored at least every six months.Efficacy is determined by a significant reduction in progression of atreatment group relative to a placebo group.

A second phase II trial is performed to evaluate conversion of patientsfrom non-Alzheimer's Disease early memory loss, sometimes referred to asage-associated memory impairment (AAMI), or mild cognitive impairment(MCI), to probable Alzheimer's disease as defined as by ADRDA criteria.Patients with high risk for conversion to Alzheimer's Disease areselected from a non-clinical population by screening referencepopulations for early signs of memory loss or other difficultiesassociated with pre-Alzheimer's symptomatology, a family history ofAlzheimer's Disease, genetic risk factors, age, sex, and other featuresfound to predict high-risk for Alzheimer's Disease. Baseline scores onsuitable metrics including the MMSE and the ADAS together with othermetrics designed to evaluate a more normal population are collected.These patient populations are divided into suitable groups with placebocomparison against dosing alternatives with the agent. These patientpopulations are followed at intervals of about six months, and theendpoint for each patient is whether or not he or she converts toprobable Alzheimer's Disease as defined by ADRDA criteria at the end ofthe observation.

XIII. General Materials and Methods

1. Measurement of Antibody Titers

Mice were bled by making a small nick in the tail vein and collectingabout 200 μl of blood into a microfuge tube. Guinea pigs were bled byfirst shaving the back hock area and then using an 18 gauge needle tonick the metatarsal vein and collecting the blood into microfuge tubes.Blood was allowed to clot for one hr at room temperature (RT), vortexed,then centrifuged at 14,000×g for 10 min to separate the clot from theserum. Serum was then transferred to a clean microfuge tube and storedat 4° C. until titered.

Antibody titers were measured by ELISA. 96-well microtiter plates(Costar EIA plates) were coated with 100 μl of a solution containingeither 10 μg/ml either Aβ42 or SAPP or other antigens as noted in eachof the individual reports in Well Coating Buffer (0.1 M sodiumphosphate, pH 8.5, 0.1% sodium azide) and held overnight at RT. Thewells were aspirated and sera were added to the wells starting at a1/100 dilution in Specimen Diluent (0.014 M sodium phosphate, pH 7.4,0.15 M NaCl, 0.6% bovine serum albumin, 0.05% thimerosal). Seven serialdilutions of the samples were made directly in the plates in three-foldsteps to reach a final dilution of 1/218,700. The dilutions wereincubated in the coated-plate wells for one hr at RT. The plates werethen washed four times with PBS containing 0.05% Tween 20. The secondantibody, a goat anti-mouse Ig conjugated to horseradish peroxidase(obtained from Boehringer Mannheim), was added to the wells as 100 μl ofa 1/3000 dilution in Specimen Diluent and incubated for one hr at RT.Plates were again washed four times in PBS, Tween 20. To develop thechromogen, 100 μl of Slow TMB (3,340 ,5,5′-tetramethyl benzidineobtained from Pierce Chemicals) was added to each well and incubated for15 min at RT. The reaction was stopped by the addition of 25 μl of 2 MH₂SO₄. The color intensity was then read on a Molecular Devices Vmax at(450 nm-650 nm).

Titers were defined as the reciprocal of the dilution of serum givingone half the maximum OD. Maximal OD was generally taken from an initial1/100 dilution, except in cases with very high titers, in which case ahigher initial dilution was necessary to establish the maximal OD. Ifthe 50% point fell between two dilutions, a linear extrapolation wasmade to calculate the final titer. To calculate geometric mean antibodytiters, titers less than 100 were arbitrarily assigned a titer value of25.

2. Lymphocyte Proliferation Assay

Mice were anesthetized with isoflurane. Spleens were removed and rinsedtwice with 5 ml PBS containing 10% heat-inactivated fetal bovine serum(PBS-FBS) and then homogenized in a 50° Centricon unit (Dako A/S,Denmark) in 1.5 ml PBS-FBS for 10 sec at 100 rpm in a Medimachine (Dako)followed by filtration through a 100 micron pore size nylon mesh.Splenocytes were washed once with 15 ml PBS-FBS, then pelleted bycentrifugation at 200×g for 5 min. Red blood cells were lysed byresuspending the pellet in 5 mL buffer containing 0.15 M NH4Cl, 1 MKHCO3, 0.1 M NaEDTA, pH 7.4 for five min at RT. Leukocytes were thenwashed as above. Freshly isolated spleen cells (10⁵ cells per well) werecultured in triplicate sets in 96-well U-bottomed tissue culture-treatedmicrotiter plates (Coming, Cambridge, Mass.) in RPMI 1640 medium (JRHBiosciences, Lenexa, Kans.) supplemented with 2.05 mM L glutamine, 1%Penicillin/Streptomycin, and 10% heat-inactivated FBS, for 96 hr at 37°C. Various A peptides, Aβ1-16, Aβ1-40, Aβ1-42 or Aβ40-1 reverse sequenceprotein were also added at doses ranging from 5 to 0.18 micromolar infour steps. Cells in control wells were cultured with Concanavalin A(Con A) (Sigma, cat. # C-5275, at 1 microgram/ml) without added protein.Cells were pulsed for the final 24 hr with 3H-thymidine (1 μCi/wellobtained from Amersham Corp., Arlington Heights Ill.). Cells were thenharvested onto UniFilter plates and counted in a Top Count MicroplateScintillation Counter (Packard Instruments, Downers Grove, Ill.).Results are expressed as counts per minute (cpm) of radioactivityincorporated into insoluble macromolecules.

4. Brain Tissue Preparation

After euthanasia, the brains were removed and one hemisphere wasprepared for immunohistochemical analysis, while three brain regions(hippocampus, cortex and cerebellum) were dissected from the otherhemisphere and used to measure the concentration of various Aβ proteinsand APP forms using specific ELISAs (Johnson-Wood et al., supra).

Tissues destined for ELISAs were homogenized in 10 volumes of ice-coldguanidine buffer (5.0 M guanidine-HCl, 50 mM Tris-HCl, pH 8.0). Thehomogenates were mixed by gentle agitation using an Adams Nutator(Fisher) for three to four hr at RT, then stored at −20° C. prior toquantitation of Aβ and APP. Previous experiments had shown that theanalytes were stable under this storage condition, and that synthetic Aβprotein (Bachem) could be quantitatively recovered when spiked intohomogenates of control brain tissue from mouse littermates (Johnson-Woodet al., supra).

5. Measurement of Aβ Levels

The brain homogenates were diluted 1:10 with ice cold Casein Diluent(0.25% casein, PBS, 0.05% sodium azide, 20 μg/ml aprotinin, 5 mM EDTA pH8.0, 10 μg/ml leupeptin) and then centrifuged at 16,000×g for 20 min at4° C. The synthetic Aβ protein standards (1-42 amino acids) and the APPstandards were prepared to include 0.5 M guanidine and 0.1% bovine serumalbumin (BSA) in the final composition. The “total” Aβ sandwich ELISAutilizes monoclonal antibody monoclonal antibody 266, specific for aminoacids 13-28 of Aβ (Seubert, et al.), as the capture antibody, andbiotinylated monoclonal antibody 3D6, specific for amino acids 1-5 of Aβ(Johnson-Wood, et al), as the reporter antibody. The 3D6 monoclonalantibody does not recognize secreted APP or full-length APP, but detectsonly Aβ species with an amino-terminal aspartic acid. This assay has alower limit of sensitivity of ˜50 ñg/ml (11 ñM) and shows nocross-reactivity to the endogenous murine Aβ protein at concentrationsup to 1 ng/ml (Johnson-Wood et al., supra).

The brain homogenates were diluted 1:10 with ice cold Casein Diluent(0.25% casein, PBS, 0.05% sodium azide, 20 μg/ml aprotinin, 5 mM EDTA pH8.0, 10 μg/ml leupeptin) and then centrifuged at 16,000×g for 20 min at4 C. The synthetic Aβ protein standards (1-42 amino acids) and the APPstandards were prepared to include 0.5 M guanidine and 0.1% bovine serumalbumin (BSA) in the final composition. The “total” Aβ sandwich ELISAutilizes monoclonal antibody (mAb) 266, specific for amino acids 13-28of Aβ (Seubert, et al.), as the capture antibody, and biotinylated mAb3D6, specific for amino acids 1-5 of Aβ (Johnson-Wood, et al), as thereporter antibody. The 3D6 mAb does not recognize secreted APP orfull-length APP, but detects only Aβ species with an amino-terminalaspartic acid. The cell line producing the antibody 3D6 has the ATCCaccession number PTA-5130, having been deposited on Apr. 8, 2003. Thisassay has a lower limit of sensitivity of ˜50 pg/ml (11 pM) and shows nocross-reactivity to the endogenous murine Aβ protein at concentrationsup to 1 ng/ml (Johnson-Wood et al., supra).

The Aβ1-42 specific sandwich ELISA employs mAβ 21F12, specific for aminoacids 33-42 of Aβ (Johnson-Wood, et al.), as the capture antibody.Biotinylated mAβ 3D6 is also the reporter antibody in this assay whichhas a lower limit of sensitivity of about 125 μg/ml (28 μM, Johnson-Woodet al.). For the Aβ ELISAs, 100 μl of either mAβ 266 (at 10 μg/ml) ormAβ 21F12 at (5 μg/ml) was coated into the wells of 96-well immunoassayplates (Costar) by overnight incubation at RT. The solution was removedby aspiration and the wells were blocked by the addition of 200 μl of0.25% human serum albumin in PBS buffer for at least 1 hr at RT.Blocking solution was removed and the plates were stored desiccated at4° C. until used. The plates were rehydrated with Wash Buffer[Tris-buffered saline (0.15 M NaCl, 0.01 M Tris-HCl, pH 7.5), plus 0.05%Tween 20] prior to use. The samples and standards were added intriplicate aliquots of 100 μl per well and then incubated overnight at4° C. The plates were washed at least three times with Wash Bufferbetween each step of the assay. The biotinylated mAβ 3D6, diluted to 0.5μg/ml in Casein Assay Buffer (0.25% casein, PBS, 0.05% Tween 20, pH7.4), was added and incubated in the wells for 1 hr at RT. Anavidin-horseradish peroxidase conjugate, (Avidin-HRP obtained fromVector, Burlingame, Calif.), diluted 1:4000 in Casein Assay Buffer, wasadded to the wells for 1 hr at RT. The colorimetric substrate, SlowTMB-ELISA (Pierce), was added and allowed to react for 15 minutes at RT,after which the enzymatic reaction was stopped by the addition of 25 μl2 N H2SO4. The reaction product was quantified using a Molecular DevicesVmax measuring the difference in absorbance at 450 nm and 650 nm.

6. Measurement of APP Levels

Two different APP assays were utilized. The first, designated APP-α/FL,recognizes both APP-alpha (α) and full-length (FL) forms of APP. Thesecond assay is specific for APP-α. The APP-α/FL assay recognizessecreted APP including the first 12 amino acids of Aβ. Since thereporter antibody (2H3) is not specific to the α-clip-site, occurringbetween amino acids 612-613 of APP695 (Esch et al., Science 248,1122-1124 (1990)); this assay also recognizes full length APP (APP-FL).Preliminary experiments using immobilized APP antibodies to thecytoplasmic tail of APP-FL to deplete brain homogenates of APP-FLsuggest that approximately 30-40% of the APP-α/FL APP is FL (data notshown). The capture antibody for both the APP-α/FL and APP-α assays ismAb 8E5, raised against amino acids 444 to 592 of the APP695 form (Gameset al., supra). The reporter mAb for the APP-α/FL assay is mAb 2H3,specific for amino acids 597-608 of APP695 (Johnson-Wood et al., supra)and the reporter antibody for the APP-α assay is a biotinylatedderivative of mAb 16H9, raised to amino acids 605 to 611 of APP. Thelower limit of sensitivity of the APP-αFL assay is about 11 ng/ml (150ρM) (Johnson-Wood et al.) and that of the APP-α specific assay is 22ng/ml (0.3 nM). For both APP assays, mAb 8E5 was coated onto the wellsof 96-well EIA plates as described above for mAb 266. Purified,recombinant secreted APP-α was used as the reference standard for theAPP-α assay and the APP-α/FL assay (Esch et al., supra). The brainhomogenate samples in 5 M guanidine were diluted 1:10 in ELISA SpecimenDiluent (0.014 M phosphate buffer, pH 7.4, 0.6% bovine serum albumin,0.05% thimerosal, 0.5 M NaCl, 0.1% NP40). They were then diluted 1:4 inSpecimen Diluent containing 0.5 M guanidine. Diluted homogenates werethen centrifuged at 16,000×g for 15 seconds at RT. The APP standards andsamples were added to the plate in duplicate aliquots and incubated for1.5 hr at RT. The biotinylated reporter antibody 2H3 or 16H9 wasincubated with samples for 1 hr at RT. Streptavidin-alkaline phosphatase(Boehringer Mannheim), diluted 1:1000 in specimen diluent, was incubatedin the wells for 1 hr at RT. The fluorescent substrate4-methyl-umbellipheryl-phosphate was added for a 30-min RT incubationand the plates were read on a Cytofluor tm 2350 fluorimeter (Millipore)at 365 nm excitation and 450 nm emission.

7. Immunohistochemistry

Brains were fixed for three days at 4° C. in 4% paraformaldehyde in PBSand then stored from one to seven days at 4° C. in 1% paraformaldehyde,PBS until sectioned. Forty-micron-thick coronal sections were cut on avibratome at RT and stored in cryoprotectant (30% glycerol, 30% ethyleneglycol in phosphate buffer) at −20° C. prior to immunohistochemicalprocessing. For each brain, six sections at the level of the dorsalhippocampus, each separated by consecutive 240 μm intervals, wereincubated overnight with one of the following antibodies: (1) abiotinylated anti-Aβ (mAb, 3D6, specific for human Aβ) diluted to aconcentration of 2 μg/ml in PBS and 1% horse serum; or (2) abiotinylated mAb specific for human APP, 8E5, diluted to a concentrationof 3 μg/ml in PBS and 1.0% horse serum; or (3) a mAb specific for glialfibrillary acidic protein (GFAP; Sigma Chemical Co.) diluted 1:500 with0.25% Triton X-100 and 1% horse serum, in Tris-buffered saline, pH 7.4(TBS); or (4) a mAb specific for CD11b, MAC-1 antigen, (ChemiconInternational) diluted 1:100 with 0.25% Triton X-100 and 1% rabbit serumin TBS; or (5) a mAb specific for MHC II antigen, (Pharmingen) diluted1:100 with 0.25% Triton X-100 and 1% rabbit serum in TBS; or (6) a ratmAb specific for CD 43 (Pharmingen) diluted 1:100 with 1% rabbit serumin PBS or (7) a rat mAb specific for CD 45RA (Pharmingen) diluted 1:100with 1% rabbit serum in PBS; or (8) a rat monoclonal Aβ specific for CD45RB (Pharmingen) diluted 1:100 with 1% rabbit serum in PBS; or (9) arat monoclonal Aβ specific for CD 45 (Pharmingen) diluted 1:100 with 1%rabbit serum in PBS; or (10) a biotinylated polyclonal hamster Aβspecific for CD3e (Pharmingen) diluted 1:100 with 1% rabbit serum in PBSor ( 11) a rat mAb specific for CD3 (Serotec) diluted 1:200 with 1%rabbit serum in PBS; or with (12) a solution of PBS lacking a primaryantibody containing 1% normal horse serum.

Sections reacted with antibody solutions listed in 1,2 and 6-12 abovewere pretreated with 1.0% Triton X-100, 0.4% hydrogen peroxide in PBSfor 20 min at RT to block endogenous peroxidase. They were nextincubated overnight at 4° C. with primary antibody. Sections reactedwith 3D6 or 8E5 or CD3e mAbs were then reacted for one hr at RT with ahorseradish peroxidase-avidin-biotin-complex with kit components “A” and“B” diluted 1:75 in PBS (Vector Elite Standard Kit, Vector Labs,Burlingame, Calif.). Sections reacted with antibodies specific for CD45RA, CD 45RB, CD 45, CD3 and the PBS solution devoid of primaryantibody were incubated for 1 hour at RT with biotinylated anti-rat IgG(Vector) diluted 1:75 in PBS or biotinylated anti-mouse IgG (Vector)diluted 1:75 in PBS, respectively. Sections were then reacted for one hrat RT with a horseradish peroxidase-avidin-biotin-complex with kitcomponents “A” and “B” diluted 1:75 in PBS (Vector Elite Standard Kit,Vector Labs, Burlingame, Calif.).

Sections were developed in 0.01% hydrogen peroxide, 0.05%3,3′-diaminobenzidine (DAB) at RT. Sections destined for incubation withthe GFAP-, MAC-1-AND MHC II-specific antibodies were pretreated with0.6% hydrogen peroxide at RT to block endogenous peroxidase thenincubated overnight with the primary antibody at 4° C. Sections reactedwith the GFAP antibody were incubated for 1 hr at RT with biotinylatedanti-mouse IgG made in horse (Vector Laboratories; Vectastain Elite ABCKit) diluted 1:200 with TBS. The sections were next reacted for one hrwith an avidin-biotin-peroxidase complex (Vector Laboratories;Vectastain Elite ABC Kit) diluted 1:1000 with TBS. Sections incubatedwith the MAC-1-or MHC II-specific monoclonal antibody as the primaryantibody were subsequently reacted for 1 hr at RT with biotinylatedanti-rat IgG made in rabbit diluted 1:200 with TBS, followed byincubation for one hr with avidin-biotin-peroxidase complex diluted1:1000 with TBS. Sections incubated with GFAP-, MAC-1- and MHCII-specific antibodies were then visualized by treatment at RT with0.05% DAB, 0.01% hydrogen peroxide, 0.04% nickel chloride, TBS for 4 and11 min, respectively.

Immunolabeled sections were mounted on glass slides (VWR, Superfrostslides), air dried overnight, dipped in Propar (Anatech) and overlaidwith coverslips using Permount (Fisher) as the mounting medium.

To counterstain Aβ plaques, a subset of the GFAP-positive sections weremounted on Superfrost slides and incubated in aqueous 1% Thioflavin S(Sigma) for 7 min following immunohistochemical processing. Sectionswere then dehydrated and cleared in Propar, then overlaid withcoverslips mounted with Permount.

8. Image Analysis

A Videometric 150 Image Analysis System (Oncor, Inc., Gaithersburg, Md.)linked to a Nikon Microphot-FX microscope through a CCD video camera anda Sony Trinitron monitor was used for quantification of theimmunoreactive slides. The image of the section was stored in a videobuffer and a color-and saturation-based threshold was determined toselect and calculate the total pixel area occupied by the immunolabeledstructures. For each section, the hippocampus was manually outlined andthe total pixel area occupied by the hippocampus was calculated. Thepercent amyloid burden was measured as: (the fraction of the hippocampalarea containing Aβ deposits immunoreactive with mAb 3D6)×100. Similarly,the percent neuritic burden was measured as: (the fraction of thehippocampal area containing dystrophic neurites reactive with monoclonalantibody 8E5)×100. The C-Imaging System (Compix, Inc., CranberryTownship, Pa.) operating the Simple 32 Software Application program waslinked to a Nikon Microphot-FX microscope through an Optronics cameraand used to quantitate the percentage of the retrospenial cortexoccupied by GFAP-positive astrocytes and MAC-1-and MHC II-positivemicroglia. The image of the immunoreacted section was stored in a videobuffer and a monochrome-based threshold was determined to select andcalculate the total pixel area occupied by immunolabeled cells. For eachsection, the retrosplenial cortex (RSC) was manually outlined and thetotal pixel area occupied by the RSC was calculated. The percentastrocytosis was defined as: (the fraction of RSC occupied byGFAP-reactive astrocytes)×100. Similarly, percent microgliosis wasdefined as: (the fraction of the RSC occupied by MAC-1- or MHCII-reactive microglia)×100. For all image analyses, six sections at thelevel of the dorsal hippocampus, each separated by consecutive 240 μmintervals, were quantitated for each animal. In all cases, the treatmentstatus of the animals was unknown to the observer.

Although the foregoing invention has been described in detail forpurposes of clarity of understanding, it will be obvious that certainmodifications may be practiced within the scope of the appended claims.All publications and patent documents cited herein are herebyincorporated by reference in their entirety for all purposes to the sameextent as if each were so individually denoted.

1-163. (canceled)
 164. A method to inhibit the formation of amyloidplaques in humans, comprising administering to a human subject in needof such inhibition an effective amount of a humanized antibody orfragment thereof that specifically immunoreacts with an epitopecontained in positions 13-28 of Aβ.
 165. A method to reduce amyloidplaques in humans, comprising administering to a human subject in needof such reduction an effective amount of a humanized antibody orfragment thereof which specifically immunoreacts with an epitopecontained in positions 13-28 of Aβ.
 166. A method to inhibit theformation of amyloid plaques in humans, comprising administering to ahuman subject in need of such inhibition an effective amount of ahumanized antibody or fragment thereof that binds to soluble Aβ peptide.167. A method to inhibit the formation of amyloid plaques in humans,comprising administering to a human subject in need of such inhibitionan effective amount of a humanized antibody or fragment thereof thatsequesters Aβ peptide from its bound, circulating form in blood.
 168. Amethod to reduce amyloid plaques in humans, comprising administering toa human subject in need of such reduction an effective amount of ahumanized antibody or fragment thereof which binds to soluble Aβpeptide.
 169. A method to reduce amyloid plaques in humans, comprisingadministering to a human subject in need of such reduction an effectiveamount of a humanized antibody or fragment thereof which sequesters Aβpeptide from its bound, circulating form in blood.
 170. The method ofany one of claims 165-169, wherein the subject is diagnosed withclinical or pre-clinical Alzheimer's disease or Down's syndrome. 171.The method of any one of claims 170, wherein the antibody isadministered by a peripheral route.
 172. The method of claim 171,wherein the antibody is administered by an oral, intraperitoneal,subcutaneous, intramuscular, or intravenous route.
 173. The method ofany one of claims 164-169, wherein the subject is not diagnosed withclinical or pre-clinical Alzheimer's disease or Down's syndrome. 174.The method of claim 173, wherein the antibody is administered by aperipheral route.
 175. The method of claim 174, wherein the antibody isadministered by an oral, intraperitoneal, subcutaneous, intramuscular,or intravenous route.
 176. The method of any one of claims 164-169,wherein the antibody or fragment, when administered peripherally tohumans, does not need to cross the blood-brain barrier to inhibit theformation of amyloid plaques.
 177. The method of claim 176, wherein thesubject is diagnosed with clinical or pre-clinical Alzheimer's diseaseor Down's syndrome.
 178. The method of claim 177, wherein the antibodyis administered by a peripheral route.
 179. The method of claim 178,wherein the antibody is administered by an oral, intraperitoneal,subcutaneous, intramuscular, or intravenous route.
 180. The method ofclaim 176, wherein the subject is not diagnosed with clinical orpre-clinical Alzheimer's disease or Down's syndrome.
 181. The method ofclaim 180, wherein the antibody is administered by a peripheral route.182. The method of claim 181, wherein the antibody is administered by anoral, intraperitoneal, subcutaneous, intramuscular, or intravenousroute.
 183. The method of any one of claims 164-169, wherein theantibody or fragment, when administered peripherally to humans, does notelicit cellular responses to inhibit the formation of amyloid plaques.184. The method of claim 183, wherein the subject is diagnosed withclinical or pre-clinical Alzheimer's disease or Down's syndrome. 185.The method of claim 184, wherein the antibody is administered by aperipheral route.
 186. The method of claim 185, wherein the antibody isadministered by an oral, intraperitoneal, subcutaneous, intramuscular,or intravenous route.
 187. The method of claim 183, wherein the subjectis not diagnosed with clinical or pre-clinical Alzheimer's disease orDown's syndrome.
 188. The method of claim 187, wherein the antibody isadministered by a peripheral route.
 189. The method of claim 188,wherein the antibody is administered by an oral, intraperitoneal,subcutaneous, intramuscular, or intravenous route.
 190. The method ofany of claims 165-169, wherein the antibody or fragment, whenadministered peripherally to humans, does not substantially bindaggregated Aβ in the brain.
 191. The method of claim 190, wherein thesubject is diagnosed with clinical or pre-clinical Alzheimer's diseaseor Down's syndrome.
 192. The method of claim 191, wherein the antibodyis administered by a peripheral route.
 193. The method of claim 192,wherein the antibody is administered by an oral, intraperitoneal,subcutaneous, intramuscular, or intravenous route.
 194. The method ofclaim 190, wherein the subject is not diagnosed with clinical orpre-clinical Alzheimer's disease or Down's syndrome.
 195. The method ofclaim 194, wherein the antibody is administered by a peripheral route.196. The method of claim 195, wherein the antibody is administered by anoral, intraperitoneal, subcutaneous, intramuscular, or intravenousroute.